Involvement of nitrergic neurons in colonic motility in a rat model of ulcerative colitis.

BACKGROUND: The mechanisms underlying gastrointestinal (GI) dysmotility with ulcerative colitis (UC) have not been fully elucidated. The enteric nervous system (ENS) plays an essential role in the GI motility. As a vital neurotransmitter in the ENS, the gas neurotransmitter nitric oxide (NO) may impact the colonic motility. In this study, dextran sulfate sodium (DSS)-induced UC rat model was used for investigating the effects of NO by examining the effects of rate-limiting enzyme nitric oxide synthase (NOS) changes on the colonic motility as well as the role of the ENS in the colonic motility during UC. AIM: To reveal the relationship between the effects of NOS expression changes in NOS-containing nitrergic neurons and the colonic motility in a rat UC model. METHODS: Male rats (n = 8/each group) were randomly divided into a control (CG), a UC group (EG1), a UC + thrombin derived polypeptide 508 trifluoroacetic acid (TP508TFA; an NOS agonist) group (EG2), and a UC + NG-monomethyl-L-arginine monoacetate (L-NMMA; an NOS inhibitor) group (EG3). UC was induced by administering 5.5% DSS in drinking water without any other treatment (EG1), while the EG2 and EG3 were gavaged with TP508 TFA and L-NMMA, respectively. The disease activity index (DAI) and histological assessment were recorded for each group, whereas the changes in the proportion of colonic nitrergic neurons were counted using immunofluorescence histochemical staining, Western blot, and enzyme linked immunosorbent assay, respectively. In addition, the contractile tension changes in the circular and longitudinal muscles of the rat colon were investigated in vitro using an organ bath system. RESULTS: The proportion of NOS-positive neurons within the colonic myenteric plexus (MP), the relative expression of NOS, and the NOS concentration in serum and colonic tissues were significantly elevated in EG1, EG2, and EG3 compared with CG rats. In UC rats, stimulation with agonists and inhibitors led to variable degrees of increase or decrease for each indicator in the EG2 and EG3. When the rats in EGs developed UC, the mean contraction tension of the colonic smooth muscle detected in vitro was higher in the EG1, EG2, and EG3 than in the CG group. Compared with the EG1, the contraction amplitude and mean contraction tension of the circular and longitudinal muscles of the colon in the EG2 and EG3 were enhanced and attenuated, respectively. Thus, during UC, regulation of the expression of NOS within the MP improved the intestinal motility, thereby favoring the recovery of intestinal functions. CONCLUSION: In UC rats, an increased number of nitrergic neurons in the colonic MP leads to the attenuation of colonic motor function. To intervene NOS activity might modulate the function of nitrergic neurons in the colonic MP and prevent colonic motor dysfunction. These results might provide clues for a novel approach to alleviate diarrhea symptoms of UC patients.

Role of neutrophils in a mouse model of halothane-induced liver injury.

Drug-induced liver injury (DILI) is a major safety concern in drug development. Its prediction and prevention have been hindered by limited knowledge of the underlying mechanisms, in part the result of a lack of animal models. We developed a mouse model of halothane-induced liver injury and characterized the mechanisms accounting for tissue damage. Female and male Balb/c, DBA/1, and C57BL/6J mice were injected intraperitoneally with halothane. Serum levels of alanine aminotransferase and histology were evaluated to determine liver injury. Balb/c mice were found to be the most susceptible strain, followed by DBA/1, with no significant hepatotoxicity observed in C57BL/6J mice. Female Balb/c and DBA/1 mice developed more severe liver damage compared with their male counterparts. Bioactivation of halothane occurred similarly in all three strains based on detection of liver proteins adducted by the reactive metabolite. Mechanistic investigations revealed that hepatic message levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta); IL-6, and IL-8 were significantly higher in halothane-treated Balb/c mice compared to DBA/1 and C57BL/6J mice. Moreover, a higher number of neutrophils were recruited into the liver of Balb/c mice upon halothane treatment compared with DBA/1, with no obvious neutrophil infiltration detected in C57BL/6J mice. Neutrophil depletion experiments demonstrated a crucial role for these cells in the development of halothane-induced liver injury. The halothane-initiated hepatotoxicity and innate immune response-mediated escalation of tissue damage are consistent with events that occur in many cases of DILI. In conclusion, our model provides a platform for elucidating strain-based and gender-based susceptibility factors in DILI development.

Analysis of eluted peptides from type 1 diabetes-susceptible HLA class II molecules identified novel islet protein, heparin/heparan sulfate-interacting protein.

Identification of peptides derived from pancreatic islet and presented by type 1 diabetes-susceptible MHC class II molecules has great significance to elucidate the pathogenesis of type 1 diabetes. A bulk culture of Epstein-Barr virus-transformed B-cells, which were established from a 22-year-old type 1 diabetic woman with HLA-DR4 and -DQw8, was pulsed with the homogenate of a human embryonic pancreas-derived cell line 1B2C6, and another culture was not pulsed with antigen. Peptide fractions were obtained by treatment of affinity-purified HLA-DR and -DQ molecules with 0.1% trifluoroacetic acid, and were subjected to reverse-phase high performance liquid chromatography (RP-HPLC). The RP-HPLC profiles of peptides derived from DR molecules revealed three peaks that specifically appeared after pulsing, but no such peaks were obtained from DQ molecules. From one of these three peaks, a peptide that consisted of 14 amino acids (AKSXNHTXXNQXRK, where X represents the undetermined amino acids) was identified. This peptide was derived from heparin/heparan sulfate-interacting protein (HIP). Immunostaining of pancreatic sections using antiserum for HIP peptide revealed exclusive staining of the islets. Thus, HIP was identified as an islet protein naturally processed and presented by HLA-DR4 molecules.

Members of the histone deacetylase superfamily differ in substrate specificity towards small synthetic substrates.

Histone deacetylases (HDACs) are important enzymes for the transcriptional regulation of gene expression in eukaryotic cells. Deacetylation of epsilon-acetyl-lysine residues within the N-terminal tail of core histones mediates changes in both histone-DNA and histone-non-histone protein interactions. However, surprisingly little is known about the substrate specificities of different HDACs. Here, we use the epsilon-acyl moieties of epsilon-modified l-lysine in peptidic substrates as a probe to examine the active site cavity of HDACs and HDAC-like enzymes. Measurements were based on a fluorogenic assay with small synthetic substrates. Four different enzyme preparations were used derived from rat, human, and bacterial sources. None of the enzymes was able to utilize substrates with epsilon-acyl moieties larger than acetyl, except rat liver HDAC, which was the only enzyme to convert a substrate containing epsilon-propionyl-l-lysine. All enzymes exhibited a distinct enantioselectivity toward l-lysine-containing substrates except FB188 HDAH which also deacetylated Boc-d-Lys(epsilon-acetyl)-MCA. Moreover, all enzymes also exhibited a distinct specificity for the length of the lysine side chain; acetylated ornithine, which comprises one CH(2) unit less in the side chain, was not a substrate. In line with these results, only acetylcadaverin the metabolic degradation product of lysine but neither acetylputrescine (degradation product of ornithine) nor acetylspermidine strongly inhibited enzyme activity. Boc-l-Lys(epsilon-trifluoroacetyl)-MCA was observed to be a superior substrate for FB188 HDAH, Pseudomonas aeruginosa HDAH (PA3774), and particularly HDAC 8 compared to rat liver HDAC, and is the first suitable (synthetic) substrate for (human-derived) HDAC 8 reported to date. Altogether, the results reveal clear differences in substrate specificity between different HDACs as analyzed in the fluorogenic HDAC assay. Finally, we present the first candidates for HDAC-type-selective substrates that may be useful as biochemical tools to establish the function of particular pathways and to elucidate the role of distinct HDAC subtypes in cellular differentiation and cancer.

Effects of HCFC-123 exposure to maternal and infant rhesus monkeys on hepatic biochemistry, lactational parameters and postnatal growth.

Peroxisome proliferators are a class of nongenotoxic rodent hepatocarcinogens that cause peroxisome proliferation and liver tumors when administered to rats and mice; but other species, including guinea pigs, dogs, and primates are less sensitive or refractory to the induction of peroxisome proliferation. Therefore, rodent peroxisome proliferators are not believed to pose a hepatocarcinogenic hazard to humans. Some peroxisome proliferators produce developmental toxicity in rats that is expressed as suppressed postnatal growth. To evaluate the relevance of the rat developmental effect to primates, groups of 4 lactating female Rhesus monkeys and their infants were exposed for 6 h/day, 7 days/week for 3 weeks to air or 1000 ppm HCFC-123. Animals were evaluated for clinical signs, body weights, clinical pathology parameters, and biochemical and pathological evaluations of liver biopsy samples. The effect of HCFC-123 exposure on milk quality (protein and fat concentration) was evaluated. The concentrations of HCFC-123 and the major metabolite, trifluoroacetic acid (TFA), were measured in the blood of the mothers and infants and in the milk. Exposure of monkeys to 1000 ppm HCFC-123 did not result in exposure-related clinical observations, or changes in body weight, appetence and behavior. There were no exposure-related effects on serum triglycerides, cholesterol, or glucose levels. HCFC-123 and TFA were present in milk, although maternal HCFC-123 exposure did not affect milk protein and fat content. In general, HCFC-123 was not detected in maternal or infant blood. TFA was detected in the majority of the mothers and TFA levels in infants ranged from 2 to 6 times higher than levels in the corresponding maternal blood. A pharmacokinetic analysis in a maternal animal indicated a peak concentration of TFA at approximately 1 h post-exposure, with a half-life of approximately 20 h. Liver microsomal P450 and peroxisome oxidase activities showed exposure-related decreases in CYP4A1 and CYP2E1 and acyl-CoA oxidase for animals exposed to HCFC-123. Microscopic evaluation of maternal liver from HCFC-123 exposed animals revealed mild to moderate centrilobular hepatocyte vacuolation, trace to mild centrilobular necrosis, and trace to mild subacute inflammation. The histopathological damage and altered hepatic biochemical activities produced by HCFC-123 in monkeys are not consistent with the HCFC-123 peroxisome proliferation response observed in rat livers. These findings demonstrate that HCFC-123 is not a peroxisome proliferator in adult Rhesus monkeys and postnatal exposure to HCFC-123 does not affect body weight of nursing infant monkeys.

19F NMR of trifluoroacetyl-labeled cysteine mutants of myoglobin: structural probes of nitric oxide bound to the H93G cavity mutant.

Nitric oxide (NO) binds to the myoglobin (Mb) cavity mutant, H93G, forming either a 5- or 6-coordinate Fe--NO heme complex. The H93G mutation replaces the proximal histidine of Mb with glycine, allowing exogenous ligands to occupy the proximal binding site. In the absence of the covalently attached proximal ligand, NO could bind to H93G from the proximal side of the heme rather than the typical diatomic binding pocket on the distal side when the 5-coordinate complex forms. The question of whether NO binds on the distal or proximal side was addressed by (19)F NMR. Site-directed mutagenesis was used to introduce unique cysteine residues at the protein surface on either the distal (S58C) or proximal (L149C) side, approximately equidistant from and perpendicular to the heme plane of both wild-type and H93G Mb. The cysteine thiols were alkylated with 3-bromo-1,1,1-trifluoroacetone to attach a trifluoroacetyl group at the mutation site. (19)F NMR spectra of 5-coordinate, NO bound S58C/H93G and L149C/H93G double mutants depict peaks with line widths of 100 and 23 Hz, respectively. As fluorine peaks broaden with increasing proximity to paramagnetic centers, such as 5-coordinate Fe--NO, the (19)F NMR data are consistent with NO binding in the distal heme pocket of H93G, even in the absence of a sixth axial ligand. Additionally, (19)F NMR spectra are reported for deoxy, oxy, CO, met CN, and met H(2)O forms of the labeled cysteine mutants. These results demonstrate that the fluorine probes are sensitive to subtle conformational changes in the protein structure due to ligation and oxidation state changes of the heme iron in Mb.

Modification of cysteine.

This unit describes a number of methods for modifying cysteine residues of proteins and peptides by reduction and alkylation procedures. A general procedure for alkylation of cysteine residues in a protein of known size and composition with haloacyl reagents or N-ethylmaleimide (NEM) is presented, and alternate protocols describe similar procedures for use when the size and composition are not known and when only very small amounts of protein are available. Alkylations that introduce amino groups using bromopropylamine and N-(iodoethyl)-trifluoroacetamide are also presented. Two procedures that are often used for subsequent sequence analysis of the protein, alkylation with 4-vinylpyridine and acrylamide, are described, and a specialized procedure for 4-vinylpyridine alkylation of protein that has been adsorbed onto a sequencing membrane is also presented. Reversible modification of cysteine residues by way of sulfitolysis is described, and a protocol for oxidation with performic acid for amino acid compositional analysis is also provided. Gentle oxidation of cysteine residues to disulfides by exposure to air is detailed. Support protocols are included for recrystallization of iodoacetic acid, colorimetric detection of free sulfhydryls, and desalting of modified samples.

Derivatization procedures for the detection of beta(2)-agonists by gas chromatographic/mass spectrometric analysis.

An evaluation of derivatization procedures for the detection of beta(2)-agonists is presented. The study was performed with the beta(2)-agonists bambuterol, clenbuterol, fenoterol, formoterol, salbutamol, salmeterol and terbutaline. Different derivatizating agents were employed, aiming to obtain derivatives with high selectivity to be used in the gas chromatographic/mass spectrometric analysis of beta(2)-agonists in biological samples. Trimethylsilylation was compared with different agents and the role of some catalysts was evaluated. Acylation, combined trimethylsilylation and acylation, and the formation of cyclic methylboronates were also studied. Sterical hindrance caused by different substituents at the nitrogen atom of the beta-ethanolamine lateral chain of beta(2)-agonist molecules is mainly responsible for differences in the abundances of the derivatives obtained. The use of catalysts produces an increase in the derivatization yield, especially for compounds with low steric hindrance (substituents with primary and secondary carbon atoms). The formation of trimethylsilyl (TMS) ethers is not influenced by structural molecular differences when only hydroxy groups are involved in derivatization. Combined trimethylsilylation and acylation showed that compounds with a secondary carbon atom linked to the nitrogen atom form mainly N-TFA-O-TMS derivatives, with a small amount of N-TMS-O-TMS derivatives. Compounds with tert-butyl substituents at the amino group (bambuterol, salbutamol and terbutaline) formed O-TMS derivatives as the main products, although a limited amount of trifluoroacylation at the nitrogen atom also occurred. Cyclic methylboronates were formed with bambuterol, clenbuterol, formoterol, salbutamol and salmeterol. Owing to hydroxy substituents in unsuitable positions for ring formation, this procedure was not effective for fenoterol and terbutaline. Mass spectra of different derivatives and tentative fragmentation profiles are also shown. For screening purpose (e.g. sports drug testing), derivatization with MSTFA or BSTFA alone is recommended as a comprehensive derivatization technique for beta(2)-agonists owing to minimal by-product formation; formation of cyclic methylboronates can be useful for confirmation purposes. Detection limits were obtained for the TMS and cyclic methylboronate derivatives using the derivatizing reagents MSTFA and trimethylboroxine, respectively. For most of the compounds, lower detection limits were found for the TMS derivatives.

Solution structure of a designed amphipathic antimicrobial synthetic peptide, PGAa.

A designed peptide, PGAa showed an excellent antifungal activity as well as an efficient bactericidal activity toward gram-positive, especially in the pathogenic yeast Candida albicans 28838. The solution structures of PGAa have been determined both in 40% TFE/water solution and DPC micelle by CD and NMR spectroscopy. Based on NOEs, vicinal coupling constants, backbone amide exchange rates, and chemical shift indices, PGAa formed a long amphipathic alpha-helical conformation in both TFE and DPC micelle environments, spanning the residues Ile(2)-Ala(19) in TFE and Lys(5)-Ala(19) in DPC micelle, respectively. Solution structures suggested that the hydrophobic residues would interact with the fatty acyl chains of the lipid bilayer, while the positively charged side-chains exposed to aqueous environments. Therefore, we conclude that the alpha-helical structure as well as the highly amphiphatic nature of PGAa peptide may play a critical role in its antimicrobial activity as well as selectivities in different species.

The major surface antigens of Entamoeba histolytica trophozoites are GPI-anchored proteophosphoglycans.

Trophozoites of the parasitic protozoa, Entamoeba histolytica, synthesize a cell surface lipoglycoconjugate, termed lipophosphoglycan, which is thought to be an important virulence factor and potential vaccine candidate against invasive amebiasis. Here, we show that the E. histolytica lipophosphoglycans are in fact glycosylphosphatidylinositol (GPI)-anchored proteophosphoglycans (PPGs). These PPGs contain a highly acidic polypeptide component which is rich in Asp, Glu and phosphoserine residues. This polypeptide component is extensively modified with linear glycan chains having the general structure, [Glcalpha1-6](n)Glcbeta1-6Gal (where n=2-23). These glycan chains can be released after mild-acid hydrolysis with trifluoroacetic or hydrofluoric acid and are probably attached to phosphoserine residues in the polypeptide backbone. The PPGs are further modified with a GPI anchor which differs from all other eukaryotic GPI anchors so far characterized in containing a glycan core with the structure, Gal(1)Man(2)GlcN-myo-inositol, and in being heterogeneously modified with chains of alpha-galactose. Trophozoites of the pathogenic HM-1:IMSS strain synthesize two distinct classes of PPG which have polydisperse molecular masses of 50-180 kDa (PPG-1) and 35-60 kDa (PPG-2) and are modified with glucan side-chains of different average lengths. In contrast, the non-pathogenic Rahman strain synthesizes one class of PPG which is only elaborated with short disaccharide side-chains (i.e. Glcbeta1-6Gal). However, the PPGs are abundant in all strains (8x10(7) copies per cell) and are likely to form a protective surface coat.

Analysis of amylin cleavage products provides new insights into the amyloidogenic region of human amylin.

Human amylin is the primary component of amyloid deposits found in the pancreatic beta-cells of patients with type 2 diabetes mellitus. Recently, two fragments of amylin have been identified in vivo. One fragment contains residues 17 to 37 of human amylin (AMYLIN17-37) and the other contains residues 24 to 37 (AMYLIN24-37). The secondary structure and amyloid forming ability of each peptide was determined at pH 5.5(+/-0.3) and pH 7.4(+/-0.3). Results at these two values of pH were very similar. Both peptides are predominantly unstructured in solution (CD) but adopt a significant amount of beta-sheet secondary structure upon aggregation (FTIR). Transmission electron microscopy (TEM) confirmed the presence of amyloid fibrils. AMYLIN24-37 was further dissected by studying peptides corresponding to residues 24 to 29 and 30 to 37. The AMYLIN30-37 peptide forms amyloid deposits. Samples of the 24 to 29 fragment which had TFA as the associated counterion formed ordered deposits but samples associated with HCl did not. Residues 20 to 29 are traditionally thought to be the amyloidogenic region of amylin, but this study demonstrates that peptides derived from other regions of amylin are capable of forming amyloid, and hence indicates that these regions of amylin can play a role in amyloid formation.

Peroxynitrite modification of protein thiols: oxidation, nitrosylation, and S-glutathiolation of functionally important cysteine residue(s) in the sarcoplasmic reticulum Ca-ATPase.

Skeletal muscle contraction and relaxation is efficiently modulated through the reaction of reactive oxygen-nitrogen species with sarcoplasmic reticulum protein thiols in vivo. However, the exact locations of functionally important modifications are at present unknown. Here, we determine by HPLC-MS that the modification of one (out of 24) Cys residue of the sarcoplasmic reticulum (SR) Ca-ATPase isoform SERCA1, Cys(349), by peroxynitrite is sufficient for the modulation of enzyme activity. Despite the size and nature of the SR Ca-ATPase, a 110 kDa membrane protein, identification and quantitation of Cys modification was achieved through labeling with 4-(dimethylamino)phenylazophenyl-4'-maleimide (DABMI) and/or N-(2-iodoethyl)trifluoroacetamide (IE-TFA) followed by an exhaustive tryptic digestion and on-line HPLC-UV-electrospray MS analysis. The reaction with IE-TFA generates aminoethylcysteine, a new trypsin cleavage site, which allows the production of specific peptide fragments that are diagnostic for IE-TFA labeling, conveniently identified by mass spectrometry. Exposure of the SR Ca-ATPase to low concentrations (0.1 mM) of peroxynitrite resulted in the fully reversible chemical modification of Cys at positions 344, 349, 471, 498, 525, and 614 (nitrosylation of Cys(344) and Cys(349) was seen), whereas higher concentrations of peroxynitrite (0.45 mM) additionally affected Cys residues at positions 636, 670, and 674. When the SR Ca-ATPase was exposed to 0.45 mM peroxynitrite in the presence of 5.0 mM glutathione (GSH), thiol modification became partially reversible and S-glutathiolation was detected for Cys residues at positions 344, 349, 364, 498, 525, and 614. The extent of enzyme inactivation (determined previously) quantitatively correlated with the loss of labeling efficiency (i) of a single Cys residue and (ii) of the tryptic fragment containing both Cys(344) and Cys(349). Earlier results had shown that the independent selective modification of Cys(344) is functionally insignificant [Kawakita, M., and Yamashita, T. (1987) J. Biochem. (Tokyo) 102, 103-109]. Thus, we conclude that modification of only Cys(349) is responsible for the modulation of the SR Ca-ATPase activity by peroxynitrite.

Epidemic of liver disease caused by hydrochlorofluorocarbons used as ozone-sparing substitutes of chlorofluorocarbons.

BACKGROUND: Hydrochlorofluorocarbons (HCFCs) are used increasingly in industry as substitutes for ozone-depleting chlorofluorocarbons (CFCs). Limited studies in animals indicate potential hepatotoxicity of some of these compounds. We investigated an epidemic of liver disease in nine industrial workers who had had repeated accidental exposure to a mixture of 1,1-dichloro-2,2,2-trifluoroethane (HCFC 123) and 1-chloro-1,2,2,2-tetrafluoroethane (HCFC 124). All nine exposed workers were affected to various degrees. Both compounds are metabolised in the same way as 1-bromo-1-chloro-2,2,2-trifluoroethane (halothane) to form reactive trifluoroacetyl halide intermediates, which have been implicated in the hepatotoxicity of halothane. We aimed to test whether HCFCs 123 and 124 can result in serious liver disease. METHODS: For one severely affected worker liver biopsy and immunohistochemical stainings for the presence of trifluoroacetyl protein adducts were done. The serum of six affected workers and five controls was tested for autoantibodies that react with human liver cytochrome-P450 2E1 (P450 2E1) and P58 protein disulphide isomerase isoform (P58). FINDINGS: The liver biopsy sample showed hepatocellular necrosis which was prominent in perivenular zone three and extended focally from portal tracts to portal tracts and centrilobular areas (bridging necrosis). Trifluoroacetyl-adducted proteins were detected in surviving hepatocytes. Autoantibodies against P450 2E1 or P58, previously associated with halothane hepatitis, were detected in the serum of five affected workers. INTERPRETATION: Repeated exposure of human beings to HCFCs 123 and 124 can result in serious liver injury in a large proportion of the exposed population. Although the exact mechanism of hepatotoxicity of these agents is not known, the results suggest that trifluoroacetyl-altered liver proteins are involved. In view of the potentially widespread use of these compounds, there is an urgent need to develop safer alternatives.

Kupffer cells from halothane-exposed guinea pigs carry trifluoroacetylated protein adducts.

The anesthetic, halothane, is bioactivated by the liver cytochrome P450 system to trifluoroacetyl-chloride, which can readily acylate liver protein. Covalent binding of the trifluoroacetyl moiety may result in hapten formation leading to the induction of an immune response and ultimately halothane hepatitis. In this study the presence of trifluoroacetylated-protein adducts in Kupffer cells was investigated to learn how the immune system might come in contact with the proteins. Guinea pigs were exposed to 1.0% halothane, 40% oxygen for 4 h. Kupffer cells were isolated on days 1 through 9 post-exposure, by liver perfusion and purification by elutriation. Using gel electrophoresis and Western blotting techniques, it has been demonstrated that Kupffer cells obtained from halothane-treated guinea pigs, do carry trifluoroacetyl-protein adducts as recognized by an anti-trifluoroacetyl-rabbit serum albumin antibody. Apparent molecular weights of polypeptides bound by trifluoroacetyl were of a wide range, 25-152 kDa. Bands were most prominent in the larger Kupffer cells with more appearing at lower molecular weights. Trifluoroacetyl-protein adducts were not detected in lung, spleen, lymph node or peripheral blood macrophages. This work suggests a role for Kupffer cells in the presentation of altered proteins in the liver to cells of the immune system.

Cytochrome P-4502E1-dependent formation of trifluoroacetyl adducts from halothane by transduced HepG2 cells.

E-9 cells, a HepG2 cell line that has the alcohol-inducible human cytochrome P-4502E1 (CYP2E1) cloned into its genome, was tested for its ability to produce trifluoroacetyl (TFA) adducts from the metabolism of halothane. The metabolism of halothane results in the production of TFA halide that can readily react with cellular proteins to form TFA adducts, which can be detected by antibodies directed against them. E-9 cells formed TFA adducts when incubated with halothane. The major consistent bands that were detected were of molecular weights of approximately 50, 78, and 100 kDa. The formation of these adducts was dependent on halothane concentration and time of incubation with halothane. MV-5 cells, the control cell line that has the viral vector without the P-450, did not produce any adducts. Inhibitors of CYP2E1 function, such as 4-methylpyrazole, inhibited adduct formation. Furthermore, phorbol esters, which have been shown to increase the CYP2E1 level in this cell line, increased TFA adduct formation. This HepG2 cell line may be of value in studying the metabolism and toxicity of halothane in a human cell culture model.

Metabolism of 1,1-dichloro-2,2,2-trifluoroethane in rats.

1. Chlorofluorohydrocarbons are presently being developed as alternatives for ozone-depleting chlorofluorocarbons. 1,1-Dichloro-2,2,2-trifluoro-[2-14C]-ethane (HCFC-123) is a chlorofluorohydrocarbon with potential widespread use and associated human exposure. As a part of the toxicological evaluation of HCFC-123, its metabolism was studied in rodents in a closed recirculating exposure system. 2. Two male rats were individually exposed for 6 h. Excretion of radioactivity was monitored for 48 h after the start of the exposure. Of the radioactivity introduced into the chamber, 14% was recovered in urine within the period of observation. Excretion of metabolites in the urine was very slow. 3. Trifluoroacetic acid was the major metabolite of HCFC-123 and N-trifluoroacetyl-2-aminoethanol and N-acetyl-S-(2,2-dichloro-1,1-difluoroethyl)-L-cysteine were identified as minor urinary metabolites of HCFC-123. 4. Forty-eight hours after the start of the exposure, covalent binding of radioactive metabolites to protein was highest in liver followed by kidney and lung. Covalent binding above background levels was not observed in pancreas and testis, the target organs of HCFC-123 tumourigenicity. 5. These results suggest that the biotransformation of HCFC-123 in rodents follows a pathway identical to those of the extensively studied structural analogue halothane.

Molecular mimicry of trifluoroacetylated human liver protein adducts by constitutive proteins and immunochemical evidence for its impairment in halothane hepatitis.

A monospecific antibody (anti-CF3CO antibody) was obtained by affinity chromatography on a N epsilon-trifluoroacetyl-L-lysine (CF3CO-Lys) matrix of a rabbit polyclonal antiserum, directed against trifluoroacetylated protein adducts (CF3CO-proteins). The anti-CF3CO antibody recognized distinct CF3CO-proteins on immunoblots of a liver biopsy obtained from a human individual 10 h after halothane anaesthesia. Cross-reactive proteins of 52 kDa and 64 kDa were recognized on immunoblots of livers obtained from human individuals not exposed to halothane. Recognition of both CF3CO-proteins and the 52-kDa and 64-kDa cross-reactive proteins was abolished in the presence of 1 mM CF3CO-Lys. Anti-CF3CO antibody, affinity-adsorbed to the 52-kDa or the 64-kDa cross-reactive proteins of human liver, recognized the majority of target CF3CO-proteins on immunoblots of the human liver biopsy of an individual exposed to halothane. Liver biopsies of 5 out of 7 (71%) patients with halothane hepatitis exhibited an absence or low amounts of immunorecognizable 52-kDa and/or 64-kDa cross-reactive proteins. In contrast, of 22 control human individuals tested, all liver tissue samples were positive for the 52-kDa and/or the 64-kDa cross-reactive proteins. These data indicate that epitopes on the cross-reactive proteins of 52 kDa and 64 kDa of human liver bear strong immunochemical resemblance to epitopes on human liver CF3CO-proteins. Low-level expression of the cross-reactive proteins of 52 kDa and 64 kDa is discussed as one possible factor in human susceptibility to halothane hepatitis.

Halothane metabolism: immunochemical evidence for molecular mimicry of trifluoroacetylated liver protein adducts by constitutive polypeptides.

A monoform antibody [anti-TFA antibody] against TFA-protein adducts (TFA-adducts) was obtained by affinity purification of a polyclonal antiserum, raised in rabbits against TFA-rabbit serum albumin, on a N-epsilon-TFA-L-lysine matrix coupled to Affi-Gel 102. The anti-TFA antibody did recognize TFA-adducts of distinct molecular mass on Western blots of hepatocyte homogenates or microsomal membranes obtained from rats pretreated with halothane. The anti-TFA antibody also recognized cross-reactive polypeptides with apparent molecular masses of 52 kDa and 64 kDa on Western blots of hepatocyte homogenates obtained from rats not treated with halothane or metabolites thereof. The 52-kDa and 64-kDa cross-reactive polypeptides were localized in the 3,000 x g particulate fraction of liver homogenates. Recognition, on Western blots, of TFA-adducts and both the 52-kDa and 64-kDa cross-reactive polypeptides by anti-TFA antibody was sensitive to competition by N-epsilon-TFA-L-lysine (IC50 less than 100 microM) and N-epsilon-acetyl-L-lysine (IC50 approximately 10 mM). Treatment with piperidine (1 M) did abolish the recognition of TFA-adducts but not that of the 52-kDa and the 64-kDa cross-reactive polypeptides by anti-TFA antibody on Western blots. In antibody-exchange experiments, anti-TFA antibody was affinity-adsorbed on Western blots to the 52-kDa or the 64-kDa cross-reactive polypeptides of the rat heart, followed by spontaneous transfer to target TFA-adducts present on Western blots of rat liver microsomal membranes. The majority of these target TFA-adducts were recognized by anti-TFA antibody transferring from the source 52-kDa or 64-kDa cross-reactive polypeptides. When examined up to 10 days after exposure of rats to a single dose of halothane, no influence on the constitutive level of expression, in the liver, of either cross-reactive polypeptide was observed. In contrast, TFA-adducts were persistent for greater than 90 hr but less than 10 days. In addition to the liver, the 52-kDa and the 64-kDa cross-reactive polypeptides were prominently expressed in the heart and the kidney and, to a much lesser degree, in the spleen, the thymus, the lung, and skeletal muscle of the rat. Considerable variation in the level of expression of the 52-kDa and the 64-kDa cross-reactive polypeptides was recognized in livers of the six human individuals tested so far.(ABSTRACT TRUNCATED AT 250 WORDS)

Human anti-endoplasmic reticulum antibodies in sera of patients with halothane-induced hepatitis are directed against a trifluoroacetylated carboxylesterase.

Previous studies have demonstrated that patients with halothane-induced hepatitis have serum antibodies that are directed against novel liver microsomal neoantigens and have suggested that these neoantigens may play an immunopathological role in development of the patients' liver damage. These investigations have further revealed that the antibodies are directed against distinct polypeptide fractions (100 kDa, 76 kDa, 59 kDa, 57 kDa, 54 kDa) that have been covalently modified by the reactive trifluoroacetyl halide metabolite of halothane. In this paper, the trifluoroacetylated (TFA) 59-kDa neoantigen (59-kDa-TFA) recognized by the patients' antibodies was isolated from liver microsomes of halothane-treated rats by chromatography on an immunoaffinity column of anti-TFA IgG. Antibodies were raised against the 59-kDa-TFA protein and were used to purify the native protein from liver microsomes of untreated rats. Based upon its apparent monomeric molecular mass, NH2-terminal amino acid sequence, catalytic activity, and other physical properties, the protein has been identified as a previously characterized microsomal carboxylesterase (EC 3.1.1.1). A similar strategy may be used to purify and characterized neoantigens associated with other drug toxicities that are believed to have an immunopathological basis.