Methyl palmitate: inhibitor of phagocytosis in primary rat Kupffer cells.

Kupffer cells are involved in phagocytosis and known to release biologically active mediators during early events of liver injury. Such functional properties of Kupffer cells can be modulated by methyl palmitate (MP). Therefore, efficacy of MP to modulate Kupffer cell function was evaluated in cultured primary Kupffer cells from rat liver. Phagocytic activity of Kupffer cells was measured by their capacity to phagocytize latex beads and the release of TNF-alpha, IL-10, IL-6, nitric oxide, and PGE2 was determined in cell culture medium after incubating the cells with various concentrations of MP for 24 h followed stimulation with lipopolysaccharide (LPS) for 6 h. To understand the mechanism of phagocytosis, we investigated the hydrolysis of MP, and determine ATP levels and activity of NF-kappaB in MP-inhibited Kupffer cells. A significant decrease was observed in phagocytosis. Phagocytosis evaluated at 0.5 mM MP was found to be time-dependent with a maximum decrease of 49% at 6 h. Exposure of Kupffer cells to MP followed by LPS stimulation showed a dose-dependent decrease in phagocytosis and reduced the release of TNF-alpha, IL-10, nitric oxide, and PGE2 but not of IL-6 levels in the supernatant as compared to the control. While ATP levels were unchanged, the nuclear factor NF-kappaB (p65) activity was inhibited in Kupffer cells treated with MP after LPS stimulation (35.6 RLU versus 49.6 RLU in control). Hydrolysis of MP was found to be time-dependent; maximum concentration of MP and palmitic acid (hydrolysis products) in the cell being at 3 and 6 h, respectively. In general, MP appears to reduce phagocytosis and levels of TNF-alpha, IL-10, nitric oxide, and PGE2 without affecting ATP levels and is probably mediated by NF-kappaB. This in vitro model is useful for detailed mechanistic studies of inhibition of phagocytosis by MP and other fatty acid esters.

Immunohistochemical localization of trichloroacylated protein adducts in tetrachloroethene-treated mice.

Tetrachloroethene (PCE), a common industrial solvent and environmental contaminant, is primarily used in the dry-cleaning industry. The toxicity of PCE has been linked to vision disorders, renal and hepatic cancer, and autoimmune diseases. Although the mechanism of toxicity is not fully understood, PCE forms trichloroacylated protein adducts in tissues where toxicity is known to occur. These adducts may be responsible for toxicity by altering the function of cellular proteins. Using Western blot analysis, formation of trichloroacylated protein adducts has been reported. To determine the localization of the adducts in a specific zone of a tissue, immunohistochemical staining was used in the study. An antiserum to trichloroacylated proteins was raised in rabbits and its specificity was established by enzyme-linked immunosorbent assay (ELISA). Female MRL-lpr/lpr and MRL +/+ mice were treated with PCE using a single 5-mmol/kg dose over 24 h or on every fourth day for 6 wk (total 20 doses). Formation of trichloroacylated protein adducts was observed in the liver, and localized to the centrilobular zones. Intensity and circumference of the staining around the central vein were much greater in subchronically treated mice than in acutely treated mice. No immunochemical reactivity was observed in any of the other tissues examined. This study shows that hepatic trichloroacylated protein adducts are localized in a region of the liver where PCE-mediated toxicity is known to occur. Immunohistochemical localization of these adducts and its association with PCE-induced toxicity support the contention that adducts may contribute to toxicity.

Purification and characterization of rat hepatic microsomal low molecular weight fatty acid ethyl ester synthase and its relationship to carboxylesterases.

We reported purification of a high molecular weight (HMW) (ca. 180 kD) and a low molecular weight (LMW) (ca. 60 kD) protein fractions from digitonized rat liver microsomes using ammonium sulfate precipitation followed by ion exchange and gel filtration column chromatography. Both fractions expressed fatty acid ethyl ester (FAEE) synthase as well as p-nitrophenyl acetate (PNPA)-hydrolyzing (esterase) activities. The HMW fraction was found to be a trimer with subunit molecular weight ca. 60 kD and structurally and functionally similar to rat hepatic microsomal carboxylesterase (CE, pI 6.1) and adipose tissue FAEE synthase. In this article, we report further purification and characterization of the LMW (minor) fraction expressing FAEE synthase activity and its structural and functional relationship to hepatic microsomal CEs. Using isoelectric focusing (IEF) followed by gel filtration-high-performance liquid chromatography (GF-HPLC), five proteins were purified, which expressed FAEE synthase as well as PNPA-hydrolyzing activity. The isoelectric point values of 6.5, 5.8, 5.6, 5.3, and 5.0 were found for the purified LMW proteins by IEF and each showed a peak corresponding to ca. 60 kD molecular weight by GF-HPLC, which expressed FAEE synthase as well as PNPA-hydrolyzing activity. Sodium dodecyl sulfate-polyacrylamide gel elecrophoresis (SDS-PAGE) analysis of the GF-HPLC purified LMW proteins revealed that these proteins are monomers (ca. 60 kD). All the purified LMW proteins cross-reacted with antibodies to rat adipose tissue FAEE synthase. Coelution of PNPA-hydrolyzing and FAEE synthase activity at each step of purification and cross-reactivity with rat adipose tissue FAEE synthase antibodies suggest that the purified proteins are related to various hepatic microsomal CEs. This conclusion is further supported by the homology of N-terminal amino acid sequence of the purified LMW proteins to various hepatic microsomal CEs and protease precursors. Therefore, LMW FAEE synthase activity most probably is expressed by various isozymes of hepatic microsomal CEs, which are also involved in the biotransformation of xenobiotic alcohols and amines.

Fatty acid ethyl esters and ethanol-induced pancreatitis.

Conjugation of xenobiotic alcohols with endogenous fatty acids is considered one of the mechanisms of their retention in the target organs. A number of fatty acid esters of alcohol's detected in the human tissues were found to be toxic in vivo and in vitro. Non-oxidative metabolism of ethanol resulting in the formation of fatty acid ethyl esters (FAEEs) appears to be one of the major pathways of ethanol disposition in the pancreas during chronic alcohol abuse, and could be associated with pancreatitis. In most cases, pancreatic damage occurs in alcoholics preceding the onset of clinical pancreatitis. Early markers of ethanol-induced pancreatitis could be important for early prevention of such injury. Although FAEEs have been implicated in the ethanol-induced pancreatitis, mechanism(s) of such injury is not well understood. Studies by others and by our own group have shown that plasma levels of FAEEs correlate well with plasma/blood alcohol concentration. FAEE synthase is known to catalyze the formation of FAEEs. The activity of FAEE synthase was found highest in the pancreas. Excessive synthesis of FAEEs during chronic alcohol abuse in the pancreas may be associated with pancreatic injury as supported by in vivo and cell culture studies. Human studies correlating plasma FAEE levels with that of markers of pancreatic injury could be important in developing markers of ethanol-induced toxicity. Although toxicity of exogenously administered FAEEs is shown in vivo and in vitro, the toxicity associated with endogenously formed FAEEs has not been studied. Therefore, studies regarding the role of endogenously formed FAEEs could be important in understanding the mechanism of ethanol-induced pancreatitis.

Fatty acid ethyl and methyl ester synthases, and fatty acid anilide synthase in HepG2 and AR42J cells: interrelationships and inhibition by tri-o-tolyl phosphate.

Synthesis of fatty acid ethyl esters (FAEEs), fatty acid methyl esters (FAMEs), and fatty acid anilides (FAAs) in humans and/or experimental animals and in vitro have been reported by us and other investigators. In previous studies, we have demonstrated that fatty acid ethyl ester synthase (FAEES), purified from rat liver microsomes, is structurally and functionally identical to the rat liver microsomal carboxylesterase (pI 6.1) and suggested a role in the conjugation of a variety of xenobiotic alcohols with endogenous fatty acids (B. S. Kaphalia, R. R. Fritz, and G. A. S. Ansari, Chem. Res. Toxicol. 11, 211-218, 1997). However, hepatic FAEES was found to be structurally and functionally different from that of pancreas. Therefore, the present study was undertaken to determine structural and functional interrelationships among the enzyme(s) involved in the synthesis of FAEEs, FAMEs, and FAAs, in HepG2 and AR42J cells using tri-o-tolyl phosphate (TOTP), a specific inhibitor for beta-esterases. Synthesis of FAEEs, FAMEs, and FAAs, studied in the HepG2 cells, was found to be dose- and time-dependent following incubation with methanol, ethanol, or aniline, respectively. Approximately 86-90% inhibition of FAEE, FAME, and FAA synthesizing activities was found in HepG2 cells following exposure to 2.5 microM TOTP. Identical profiles of dose- and time-dependent inhibition of FAEE, FAME, and FAA synthesizing activities by TOTP in HepG2 cells suggest that synthesis of FAEEs, FAMEs, and FAAs is catalyzed by the same enzyme(s). However, FAEE, FAME, and FAA synthesizing activities in AR42J cells could not be inhibited by TOTP under similar experimental conditions. A differential pattern of p-nitrophenyl acetate hydrolyzing activity (a measure of esterase activity) similar to that of fatty acid ester/anilide synthesizing activities was observed in the two cell lines. These results are further substantiated by the presence of approximately 60 kDa (subunit molecular weight) protein in the postnuclear fraction of HepG2 but not in AR42J cells by Western blot analysis using antibodies raised against FAEES, purified from rat liver microsomes or adipose tissue. Therefore, the enzyme responsible for the FAEE, FAME, or FAA synthesizing activities is most probably carboxylesterase in HepG2 cells and is structurally and functionally different than that present in AR42J cells. These studies also indicate the utility of HepG2 and AR42J cell cultures as an alternative to the animal model regarding studies on nonoxidative metabolism of alcohols and amines, in general.

Fatty acid anilides: in vivo formation and relevance to toxic oil syndrome.

Toxic oil syndrome (TOS), a multisystemic epidemic outbreak in 1981 in Spain, was caused by the ingestion of a cooking oil mixture containing rapeseed oil denatured with aniline. The mechanisms and causative agents responsible for the TOS are still not known. Although primary lesions observed in TOS patients could not be reproduced experimentally, the levels of fatty acid anilides (FAAs) and aniline in TOS-related cooking oil were considered proximate markers of TOS. Aniline, available from free aniline and FAAs ingested with TOS-related cooking oil, and its reconjugation with endogenous fatty acids could be an early event leading to TOS. Therefore, the present study was undertaken to determine the formation of FAAs following an oral dose of 2 mmol/kg aniline hydrochloride (AH) via gavage in rats. Here, 16:0, 18:0, 18:1, 18:2, 18:3, and 20:4 FAAs were analyzed in the whole blood, brown fat, liver, and pancreas at 0 (control), 0.25, 0.5, 1, 3, 6, 12, 24, and 48 hours. Generally, 16:0 and 18:1 FAAs were detected in the whole blood, brown fat, and liver of AH-treated rats with highest mean levels at 0.25 or 0.5 hour, except 3 hours for the whole blood. Only 16:0 FAA was detectable in the pancreas of AH-treated animals. The 18:0 FAA was also detected frequently in the liver while other FAAs were either in trace amounts or not detectable in the tissues analyzed in the present study. Overall, highest formation of the 16:0 FAA was found in the liver followed by pancreas and of 18:1 FAA in the whole blood and brown fat. These results indicate a rapid formation and further metabolism and disposition of FAAs in rat model and support our previous findings that 18:1 and 16:0 fatty acids are better substrates for the conjugation with aniline. Surprisingly, a small or trace amount of a few FAAs also detected in the tissues of control rats indicates their endogenous biosynthesis and/or presence. Results of 18:1 fatty acid incubation and aniline in the presence of fatty acid ethyl ester synthase, purified to homogeneity from rat liver microsome, suggest that formation of FAAs is catalyzed by an enzyme involved in the conjugation of fatty acids with xenobiotic alcohols. Because the FAAs are known to exert a wide range of toxicity in experimental animals and primary cell cultures, in vivo formation of FAAs could be an early event leading to TOS.

Torsional injury resulting in disc degeneration: I. An in vivo rabbit model.

Torsional injuries may be a precursor to intervertebral disc degeneration, but published rabbit models indicate a latent time of 6 months. We describe a rabbit model in which instability and disc degeneration appear within 3 months. Sixty-five male New Zealand rabbits underwent presurgical irradiation to inhibit heterotopic bone formation. Control animals then underwent either a soft-tissue release or facetectomy and capsulotomy, whereas experimental animals received surgery and an acute 30 degrees torsional lumbar injury. Capsulotomy, as well as facetectomy without torsion, failed to effect disc degeneration. However, the rabbits that received torsion exhibited clear indications of degenerative disc changes (thinning, increased PLA2 levels, and decreased nucleus pulposus volume) within 60-90 days. The observations associate disc degeneration with a destabilizing acute torsional injury.

Single step thin-layer chromatographic method for quantitation of enzymatic formation of fatty acid anilides.

The activity of the enzyme involved in catalyzing the formation of fatty acid anilides can be measured by quantitating the fatty acid anilides formed. We have shown earlier that oleic acid is the most preferred substrate among other fatty acids studied for the conjugation with aniline. The reaction product (oleyl anilide) could be separated by thin-layer chromatography (TLC) and then quantified by reversed-phase high-performance liquid chromatography (HPLC). Using [1-(14)C]oleic acid as substrate, the fatty acid anilide forming activity can be determined in a single step by TLC analysis. The conventional TLC methods used for the separation of the fatty acid esters, however, could not resolve oleyl anilide from the residual [1-(14)C]oleic acid. Therefore, a simple and reliable TLC method was developed for the separation of oleyl anilide from oleic acid using a freshly prepared solvent consisting of petroleum ether-ethyl acetate-ammonium hydroxide (80:20:1, v/v). Using this solvent system the relative flow (Rf) values were found to be 0.54 for oleyl anilide and 0.34 for aniline, whereas oleic acid remained at the origin. The TLC procedure developed in the present study could be used to determine the fatty acid anilide forming activity using [1-(14)C]oleic or other fatty acids as substrate and was also found suitable for the analysis of fatty acid anilides from the biological samples.

Acute hematopoietic toxicity of aniline in rats.

In the present study, acute hematopoietic toxicity of aniline as a function of time was investigated in rats. The animals were given a single oral dose of aniline hydrochloride (2 mmol/kg) and euthanized at zero (control), 0.25, 0.5, 1, 3, 6, 12, 24 and 48 h following the treatment. The blood methemoglobin level increased dramatically and attained a peak level of 37% (31 fold greater than the controls) at 0.5 h. Thereafter, the increases were less pronounced and the level declined with time. Spleen weight to body weight ratio remained unchanged up to 24 h, but increased approximately 25% at 48 h. Lipid peroxidation (MDA content) in the spleen increased by 39% at 24 h and remained steady even at 48 h. MDA-protein adducts, as quantitated by a competitive ELISA, showed 94, 126 and 265% increases in the spleen homogenates at 12, 24 and 48 h, respectively, following the treatment. However, no changes were observed in the splenic protein oxidation. Morphological examination showed congestion of splenic blood vessels and marked expansion of red pulp at 24 and 48 h. These studies suggest that aniline related changes in the blood are reflected very early as evident from increases in the methemoglobin content, whereas changes in the spleen appear later and are characterized by splenic weight changes, increased lipid peroxidation, MDA-protein adduct formation and morphological changes after a single high dose exposure. The increased lipid peroxidation in the spleen also suggests that free radical-mediated reactions could be the potential mechanism of splenic toxicity of aniline and lipid peroxidation precedes protein oxidation.

Time-dependent autoimmune response of dichloroacetyl chloride in female MRL +/+ mice.

Welders are exposed to dichloroacetyl chloride (DCAC) when trichloroethene (TCE) is used as a degreasing agent. Human exposure to TCE and tetrachloroethane can also lead to formation of DCAC in situ through metabolism. Due to its strong acylating property, it can bind with cellular macromolecules and act as hapten and consequently may elicit autoimmune (Al) response. Earlier, we reported that both TCE and DCAC induce/accelerate Al response in MRL +/+ mice, and DCAC even at 50 fold lower concentration induced greater Al responses. These studies, however, were conducted at a single time point (six weeks of treatment) and therefore necessitate a time-dependent characterization of this DCAC-induced Al response. Female MRL +/+ were given ip treatments of 0.2 mmol/kg DCAC in 100 microliters of corn oil every 4th day, while controls received an equal volume of corn oil only. DCAC treatment resulted in spleen weight increases at all time points whereas serum IgG showed significant increases at 4, 6 and 8 weeks of treatment. Serum autoantibodies, i.e., antinuclear antibodies, anti-single stranded DNA antibodies and anticardiolipin antibodies showed positive responses only after 4 weeks of treatment. However, the optimal responses were observed at 6 weeks and subsequently the responses diminished (at 8 weeks). The DCAC-specific antibodies showed a pattern similar to autoantibodies, i.e., an optimal response at 6 weeks of treatment. Our results thus suggest that DCAC under the current experimental conditions induces an optimal Al response at 6 weeks of treatment and further emphasize the usefulness of MRL +/+ mice in studying chemical-induced autoimmunity.

Purification and characterization of rat liver microsomal fatty acid ethyl and 2-chloroethyl ester synthase and their relationship with carboxylesterase (pI 6.1).

Previous studies have shown that fatty acid ethyl ester synthase (FAEES) which catalyzes the formation of ethyl or 2-chloroethyl esters of long-chain fatty acids is localized in the microsomal fraction of rat liver. A recent study suggests that rat adipose tissue FAEES is similar to rat liver microsomal carboxylesterase (CE) [Tsujita and Okuda (1992) J. Biol. Chem. 267, 23489-23494]. Since the interrelationships among FAEES, 2-chloroethyl ester synthase (FACEES), and cholesterol esterase (ChE) are also not clear at present, we purified and characterized FAEES and FACEES from rat hepatic microsomes and studied their functional and structural relationships with CE and ChE. The results of these studies showed that CE, FAEES, and FACEES activities copurified during each step of purification. Although gel-filtration column chromatography of DEAE-Sephacel purified microsomal protein resolved into two peaks with an estimated molecular weight of 180 (major) and 60 kDa (minor, this paper describes characterization of only the 180 kDa protein. CE, FAEES, and FACEES activities associated with homogeneous 180 kDa protein could be inhibited by a beta-esterase inhibitor (diisopropyl fluorophosphate) in an identical manner. This protein, however, showed only the hydrolytic activity, but not the synthetic activity for cholesterol oleate, indicating that it is distinct from ChE. The purified protein could be immunoprecipitated with the antibodies raised against rat adipose tissue FAEES, but not with antibodies against rat pancreatic ChE, demonstrating again that the purified protein is distinct from ChE. A single band corresponding to 60 kDa upon SDS-PAGE, under reduced denaturing conditions, indicates that the purified protein is a trimer. N-terminal amino acid sequence of the first 27 residues were identical to that of rat hepatic microsomal CE [Robbi et al. (1990) Biochem. J., 451-458] which suggests structural similarity of the purified protein with rat hepatic microsomal CE. Therefore, the functional and structural properties of the purified protein demonstrate that FAEES, FACEES, as well as CE activities are expressed by the same protein, purified in this study, which exists as a trimer (180 kDa) and is involved in biosynthesis of long-chain fatty acid esters of xenobiotic alcohols. Further studies on purification and characterization of the enzymes responsible for the esterification of xenobiotic alcohols with endogenous fatty acids from various target organs need to be conducted to determine their functional and structural interrelationships. Inhibition and induction studies of these enzyme(s) and the extent of observed toxicity could be important in understanding their role in etiology of chronic diseases induced by alcohol abuse.

Nonoxidative metabolism of 2-butoxyethanol via fatty acid conjugation in Fischer 344 rats.

Nonoxidative metabolism of ethylene glycol monobutyl ether (2-butoxyethanol or BE) via fatty acid conjugation was studied in the liver of Fischer 344 male rats following a single oral administration of 500 mg/kg body weight [ethyl-1,2-14C]BE (70 microCi/kg). Animals were killed 2 h after the treatment, hepatic lipids extracted, and the neutral lipids were separated using solid-phase extraction. The neutral lipid fraction was subjected to preparative thin-layer chromatography, and the esters corresponding to the relative flow of authentic fatty acid 2-butoxyethyl esters were recovered and analyzed by reversed-phase high-performance liquid chromatography (HPLC) using methanol-water (37:3, v/v) as solvent. Approximately 85% of the 14C label present in the ester fraction was coeluted at retention times corresponding to the different fatty acid 2-butoxyethyl ester standards. The radioactive fractions were analyzed by electron impact mass spectrometry. Molecular ion peaks and fragmentation patterns similar to that of 16:0, 18:0, 18:1, 18:2, and 20:4 fatty acid 2-butoxyethyl ester standards were detected in the corresponding radioactive HPLC fractions. Fatty acid ethyl ester synthase (FAEES), purified from the rat liver microsomal fraction, was also found to catalyze the formation of 18:1 fatty acid 2-butoxyethyl ester. These studies demonstrate that BE is metabolized nonoxidatively via conjugation with long-chain fatty acids, and the formation of these esters appears to be catalyzed by the enzyme(s) involved in fatty acid conjugation of xenobiotic alcohols. However, the biological significance of BE conjugation with fatty acids remains to be investigated.

Increased endobiotic fatty acid methyl esters following exposure to methanol.

Human exposure to methanol is likely to increase in the future due to its proposed use as an alternate automobile fuel. Since alcohols are known to esterify the fatty acids in the body and some of those esterified esters are toxic, we studied the formation of fatty acid esters of methanol in Long-Evans male rats given a single oral dose of 3.5 g/kg body weight of methanol in saline. Animals given an equal volume of saline served as control. Three rats were euthanized at 1, 3, 6, 12 and 24 hr following the treatment. Fatty acid methyl esters, extracted from whole blood, liver, pancreas, and brown fat were separated by thin-layer chromatography and quantitated by gas chromatography (GC). Their identity was then confirmed by GC-mass spectrometry. Average levels as high as 596, 5293, 2239, 1106, 9665, 7728, 562, and 2792 micrograms/g (wet weight basis) of 14:0, 16:0, 16:1, 18:0, 18:1, 18:2, 18:3, and 20:4 fatty acid methyl esters, respectively, were found in the pancreas of methanol-treated rats. The average concentration of total fatty acid methyl esters was computed to be 4513, 29594, 22871, 18956, 17014, and 9702 micrograms/g in the pancreas compared to 1.9, 25.4, 36.8, 18.5, 18.9, and 14.2 micrograms/g in the liver at 0, 1, 3, 6, 12, and 24 hr, respectively, following methanol exposure. On dry lipid weight basis, the levels were significantly higher again in pancreas followed by brown fat and liver. In whole blood, only low levels of 16:0, 18:0, and 20:4 fatty acid methyl esters could be detected at all time points. The highest concentration of total fatty acid methyl esters in the pancreas, liver, and brown fat was detected at 1, 3, and 24 hr, respectively. Most of the fatty acid methyl esters found in the liver and pancreas decreased after 6 hr of methanol exposure. The fatty acid methyl esters of higher concentrations were 16:0 in the whole blood, 18:0, 18:1, 18:2, and 20:4 in liver, 18:1, and 18:2 in pancreas and 16:0, 18:1, and 18:2 in brown fat. These fatty acid methyl esters were also detected in the tissues of control rats indicating their endogenous formation. Significant increase in methylation of the fatty acids during methanol exposure, as found in this study, may serve as a defense mechanism for preventing available methanol from oxidative metabolism to render toxicity. However, the biological significance of these fatty acid methyl esters is yet to be understood.

Trichloroethene-induced autoimmune response in female MRL +/+ mice.

Trichloroethene (TCE) has been implicated in the pathogenesis of autoimmune diseases such as systemic lupus erythematosus (SLE) and scleroderma in humans. However, experimental studies have not been conducted to establish the role of TCE in causing autoimmunity and/or SLE. To clarify the role of TCE in autoimmune responses, subchronic studies were carried out in female autoimmune prone mice (MRL +/+). Three groups of mice (5 weeks old) received intraperitoneal injections of 10 mmol/kg of TCE, 0.2 mmol/kg of dichloroacetyl chloride (DCAC) (one of the metabolites of TCE with strong acylating property), or an equal volume (100 microliters) of corn oil alone (controls). Animals were dosed every 4th day for 6 weeks and euthanized 24 hr following the last dose. Sera and major tissues were collected and analyzed. Spleen weights in the TCE and DCAC groups increased 36% with a similar pattern of change in the spleen-to-body weight ratios. Serum IgG in the TCE and DCAC groups increased 45 and 322%, respectively. Using specific ELISA assays for mice, autoimmune antibodies were detected in the sera of TCE- and DCAC-treated mice in the following patterns: for anti-nuclear antibodies; controls, 0/4; TCE, 4/4; DCAC, 3/5; for anti-ssDNA antibodies; controls, 0/4; TCE, 2/4; DCAC, 5/5; for anti-cardiolipin antibodies; controls, 0/4; TCE, 0/4; DCAC, 3/5. An ELISA developed for the measurement of DCAC-specific antibodies using conjugated DCAC-albumin as an antigen showed the following pattern: for controls, 0/4; TCE, 0/4; DCAC, 5/5. These results suggest that TCE and its metabolite, DCAC, induce and/or accelerate autoimmune responses in female MRL +/+ mice. The greater responses induced by DCAC at a dose 50 times lower than TCE suggests that this metabolite may be important in the mechanisms leading to TCE-induced autoimmunity.

Fatty acylation of the rat asialoglycoprotein receptor. The three subunits from active receptors contain covalently bound palmitate and stearate.

Rat hepatic asialoglycoprotein receptors (ASGP-Rs) are hetero-oligomers composed of three homologous glycoprotein subunits, designated rat hepatic lectins (RHL) 1, 2, and 3. ASGP-Rs mediate the endocytosis and degradation of circulating glycoconjugates containing terminal N-acetylgalactosamine or galactose, including desialylated plasma glycoproteins. We have shown in permeable rat hepatocytes that the ligand binding activity of one subpopulation of receptors (designated State 2 ASGP-Rs) can be decreased or increased, respectively, by ATP and palmitoyl-CoA (Weigel, P. H., and Oka, J. A. (1993) J. Biol. Chem. 268, 27186-27190). We proposed that a reversible and cyclic acylation/deacylation process may regulate ASGP-R activity during endocytosis, receptor-ligand dissociation, and receptor recycling. In the accompanying paper (Zeng, F-Y., and Weigel, P. H. (1995) J. Biol. Chem. 270, 21388-21395), we show that the ligand binding activity of affinity-purified State 2 ASGP-Rs is decreased by treatment with hydroxylamine under mild conditions consistent with these ASGP-Rs being fatty acylated in vivo. In this study, we used a chemical method to determine the presence of covalently-bound fatty acids in individual ASGP-R subunits. The affinity-purified ASGP-R preparations were separated by SDS-polyacrylamide gel electrophoresis under nonreducing conditions, and the gel slices containing individual RHL subunits were treated with alkali to release covalently bound fatty acids, which were subsequently analyzed by gas chromatography and confirmed by gas chromatography-mass spectrometry. Both stearic and palmitic acids were detected in all three receptor subunits. Pretreatment of ASGP-Rs with hydroxylamine before SDS-polyacrylamide gel electrophoresis reduced the content of both fatty acids by 66-80%, indicating that most of these fatty acids are attached to cysteine residues via thioester linkages. Furthermore, when freshly isolated hepatocytes were cultured in the presence of [3H]palmitate, all three RHL subunits in affinity-purified ASGP-Rs were metabolically labeled. We conclude that RHL1, RHL2, and RHL3 are modified by fatty acylation in intact cells.

Hematopoietic toxicity of linoleic acid anilide: importance of aniline.

The purpose of this study was to investigate the role of hydrolysis products of linoleic acid anilide (LAA), i.e., aniline and linoleic acid (LA), in the toxicity to the hemopoietic system, especially to the spleen. To achieve this, the parent compound (LAA) and its putative hydrolysis products, i.e., aniline or linoleic acid (LA), were given to male SD rats at equimolar doses (0.7 mmol/kg) in 0.25 ml mineral oil by gavage, daily, for 14 days. The controls received equal volumes of vehicle only. Five animals from each group were euthanized at Days 1, 7, and 28 following the last dose. At all time points, spleen weights increased in the LAA- and aniline-treated rats, but spleen to body weight ratios were increased only at Days 1 and 7 in these groups. No changes were observed in the LA-treated rats at any time point. RBC counts were decreased in the LAA and aniline groups at Days 1 and 7, whereas hemoglobin content was decreased by 20 and 13% in the LAA- and aniline-treated rats, respectively, only at Day 1. Methemoglobin content in the LAA and aniline groups also increased by 76 and 101%, respectively, at Day 1. Serum transaminases (AST and ALT) decreased in the LAA, aniline, and LA groups but the decreases were more consistent in the LA group. Serum IgA increased in the LAA and aniline groups only at Day 1. Splenic iron content was increased 381, 486, and 51% in the LAA-treated rats and 474, 491, and 58% in the aniline-treated rats at Days 1, 7, and 28, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Erythrocyte-aniline interaction leads to their accumulation and iron deposition in rat spleen.

In order to understand the splenic toxicity of aniline in rats, early interaction of aniline with erythrocytes and its subsequent deposition and covalent binding to macromolecules in target (spleen) and nontarget (liver) organs have been studied. Male Sprague-Dawley (SD) rats were given 1 or 3 doses of 1 mmol/kg [14C]aniline hydrochloride (1 dose/d) by gavage and euthanized 24 h after the treatment. Among blood components, maximum radioactivity was found to be associated with red blood cells (RBCs). After 3 doses, there was 112, 79, and 67% increase in the radioactivity in the whole blood, RBCs, and hemolysate, respectively, in comparison to 1 dose. In comparison to RBCs, plasma had only 40 and 16% radioactivity after the administration of 1 and 3 doses, respectively. Spleen homogenate at 1 dose had one-third of the radioactivity in the TCA precipitate, which increased to 40% at 3 doses, while the total radioactivity increased 256% over 1 dose. Liver, which had almost double the radioactivity on a per gram tissue basis compared to the spleen at one dose, did not show any appreciable increase in the radioactivity at three doses. However, radioactivity in the TCA precipitate of liver homogenate increased by 92% after 3 doses. The iron content of the spleen in rats given 3 doses of [14C]aniline increased by 85% compared to the rats given just 1 dose. The iron content of liver did not show any change at three doses. These data thus demonstrate a dose-dependent binding and accumulation of radioactivity in erythrocytes and spleen. These interactions, along with parallel increases in the iron content of the spleen, could be critical in the splenic toxicity of anilines.

Fatty acid conjugates of xenobiotics.

In this review, we discuss the formation and toxicity of fatty acid conjugates of xenobiotics. Conjugates formed in vivo and in vitro and those detected as contaminants are reviewed. Due to the lipophilic nature of these conjugates, they may accumulate in various body organs and cause toxic manifestations. In vivo formation of these fatty acid conjugates appears to be catalyzed by enzyme(s). Fatty acid ethyl esters are the most widely studied esters and have been implicated in the onset or pathogenesis of myocardial and pancreatic diseases in alcoholics. In experimental animals, studies on 2-chloroethyl linoleate, palmitoylpentachlorophenol and oleoyl and linoleoyl anilides clearly indicate that lipid conjugates of xenobiotics are involved in target organ toxicity. These findings warrant further detailed studies to isolate and identify other fatty acid conjugates and to evaluate their toxicity. Thorough toxicokinetic and metabolic studies are also needed to identify putative toxic agents. Identifying these agents could help in understanding the mechanism of pathogenesis associated with lipid conjugation. Finally fatty acid conjugates of drugs (prodrugs), have been shown to have increased half-lives and long-lasting dose-response. Thus these conjugates may be useful for enhancing the therapeutic potential of drugs and should be explored further.

Subchronic toxicity of aniline hydrochloride in rats.

Hematological, biochemical and histopathological responses of subchronic exposure to aniline hydrochloride (AH) have been investigated in rats. Male Sprague-Dawley rats were given 600 ppm of AH in drinking water while the control rats received tap water only. Five rats from each group were sacrificed at 30, 60, and 90 days of treatment. Organ-to-body weight ratio for spleen in the AH-treated rats was 56, 61, and 53% higher than controls at days 30, 60, and 90, respectively. Liver showed a biphasic pattern for this ratio, a decrease at 30 days and then an increase at 60 days. Among other organs, testes showed a significant decrease in this ratio at 60 days. Hematological analysis showed 65% increase in WBC counts at 30 days in the AH-treated rats, whereas, no changes were recorded at later time points. Erythrocyte counts in the AH-treated rats showed very significant decreases at all the time points, whereas, hemoglobin and hematocrit decreased at 30 and 90 days of treatment. Mean corpuscular volume and mean corpuscular hemoglobin increased in the AH-treated rats at 60 and 90 days of treatment. Methemoglobin content showed significant increases of 89, 59 and 45% at days 30, 60, and 90, respectively. Among serum immunoglobulins, IgA in the AH-treated groups showed 24 and 51% increases at days 60 and 90, respectively. Analysis of splenic lymphocyte subpopulation showed a decrease in the T-helper (CD4+/CD8-) sub-set at 90 days whereas, other subpopulations were not affected. Aniline hydroxylase activity in the liver microsomes of the AH-treated rats was significantly higher at 60 days of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Methylene dianiline: acute toxicity and effects on biliary function.

4,4'-Methylene dianiline (4,4'-diaminodiphenylmethane, DAPM), which is used in the polymer industry, causes hepatobiliary damage in exposed humans. Our objectives were to characterize the acute toxicity of DAPM in liver, particularly on secretion of biliary constituents and on biliary epithelial cell gamma-glutamyl transpeptidase (GGT) activity. Biliary cannulas were positioned in Sprague-Dawley male rats under pentobarbital anesthesia. After 1 hr of control bile collection, each rat was given 250 mg DAPM/kg (50 mg/ml) po in 35% ethanol or 35% ethanol only; bile was collected for a further 4 hr. Groups of rats were also examined for liver injury and biliary function at 8 and 24 hr after DAPM. Four hours after DAPM administration, main bile duct cells were severely damaged with minimal damage to peripheral bile ductule cells. Focal periportal hepatocellular necrosis and extensive cytolysis of cortical thymocytes occurred by 24 hr. Serum indicators of liver injury were elevated by 4 hr and continued to rise through 24 hr. By 4 hr, biliary protein concentration was increased 4-fold while concentrations of biliary bile salt, bilirubin, and glutathione were decreased by approximately 80, 50, and 200%, respectively. DAPM also induced a striking effect on biliary glucose with an approximately 20-fold increase. Histochemical staining of main bile duct GGT was absent by 8 hr after DAPM. Bile flow was diminished by 40% at 4 hr; three of five rats had no bile flow by 8 hr and none had any bile flow by 24 hr. These results indicate that DAPM rapidly diminishes bile flow and alters the secretion of biliary constituents and is highly injurious to biliary epithelial cells.