Biomedical subjects

G A Ansari

Publications and source records attributed to G A Ansari.

At least 19 recordsLinked to original sources

PubMed2004

Modulation of fatty acid methyl esters in rats pretreated with tri-o-tolyl phosphate.

Formation and toxicity of fatty acid methyl esters (FAMEs) have been reported both in vitro and in vivo. In previous studies, it was shown that fatty acid ethyl ester synthase (FAEES), which catalyzes the formation of FAMEs, also expresses esterase activity. Therefore, it was hypothesized that inhibitors of esterases such as tri-o-tolyl phosphate (TOTP) can modulate the formation of FAMEs. To test this, four groups of rats were used. Group 1 served as control (vehicle only). Group 2 was treated with methanol only (3 g/kg via gavage), group 3 was given TOTP only (100 mg/kg i.p. in corn oil), and group 4 was administered TOTP as in group 3, followed by methanol after 18 h. Three hours after exposure, animals were sacrificed and FAEES activity and FAME levels were measured in blood, liver, pancreas, and brown fat. About 95% of FAEES activity was inhibited in the liver and whole blood of TOTP-treated rats (group 3) but no inhibition was observed in the pancreas or brown fat. Total hepatic FAMEs were found to be lowest for the TOTP-treated group (3) and highest in the methanol-treated animals (group 2). Total pancreatic FAMEs in different groups were not statistically different, while significant increases were observed in the brown fat in both methanol-treated groups. To verify that the oxidative metabolism of methanol was unaffected by TOTP, alcohol dehydrogenase activity was also measured and found to be unchanged in any group as compared to control. These results demonstrate that the formation of FAMEs can be modulated in the liver and probably in blood, but not in the pancreas or brown fat by the inhibitors of FAEES.

Acyltransferases↗

PubMed2001

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.

Acylation↗

PubMed2001

Anti-malondialdehyde antibodies in MRL+/+ mice treated with trichloroethene and dichloroacetyl chloride: possible role of lipid peroxidation in autoimmunity.

Trichloroethene (TCE) and one of its metabolites dichloroacetyl chloride (DCAC) are known to induce/accelerate autoimmune (AI) response in MRL+/+ mice as evident from anti-nuclear, anti-ssDNA, anti-cardiolipin, and DCAC-specific antibodies in the serum (Khan et al., Toxicol. Appl. Pharmacol. 134, 155-160, 1995). In the present study, we measured anti-malondialdehyde antibodies (AMDA) in the serum of TCE- or DCAC-treated mice in order to understand the contribution of lipid peroxidation to this AI response. Female MRL+/+ mice (5 weeks old) received ip injections of 10 mmol/kg TCE or 0.2 mmol/kg of DCAC in corn oil (100 microl) every 4(th) day for 6 weeks, while controls received an equal volume of vehicle only, and AMDA was measured in the sera of these animals by an ELISA established in our laboratory. While TCE treatment caused only marginal induction of AMDA, DCAC treatment elicited a significant AMDA response. Furthermore, a time-response study of DCAC (0.2 mmol/kg, every 4(th) day, for 2, 4, 6, or 8 weeks) showed an induction of AMDA (3/4) after 4 weeks of treatment, which was even greater at both 6 and 8 weeks of DCAC treatment (5/5). These findings were further substantiated by the presence of AMDA in systemic lupus erythematosus-prone MRL-lpr/lpr mice as early as 6 weeks of age. Presence of AMDA, as observed in this study, not only indicates increased lipid peroxidation (oxidative stress), but also suggests a putative role of oxidative stress in inflammatory autoimmune diseases.

Acetates↗

PubMed2001

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.

Acyltransferases↗

PubMed2001

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.

Alcoholism↗

PubMed2000

Curcumin-glutathione interactions and the role of human glutathione S-transferase P1-1.

Curcumin (diferuloylmethane), a yellow pigment of turmeric with antioxidant properties has been shown to be a cancer preventative in animal studies. It contains two electrophilic alpha, beta-unsaturated carbonyl groups, which can react with nucleophilic compounds such as glutathione (GSH), but formation of the GSH-curcumin conjugates has not previously been demonstrated. In the present studies, we investigated the reactions of curcumin with GSH and the effect of recombinant human glutathione S-transferase(GST)P1-1 on reaction kinetics. Glutathionylated products of curcumin identified by FAB-MS and MALDI-MS included mono- and di-glutathionyl-adducts of curcumin as well as cyclic rearrangement products of GSH adducts of feruloylmethylketone (FMK) and feruloylaldehyde (FAL). The presence of GSTP1-1 significantly accelerated the initial rate of GSH-mediated consumption of curcumin in 10 mM potassium phosphate, pH 7.0, and 1 mM GSH. GSTP1-1 kinetics determined using HPLC indicated substrate inhibition (apparent K(m) for curcumin of 25+/-11 microM, and apparent K(i) for curcumin of 8+/-3 microM). GSTP1-1 was also shown to catalyze the reverse reaction leading to the formation of curcumin from GSH adducts of FMK and FAL.

Chromatography, High Pressure Liquid↗

PubMed2000

Contribution of nitrosobenzene to splenic toxicity of aniline.

To elucidate the mechanism(s) of splenic toxicity of aniline, studies were conducted with nitrosobenzene (NB), an N-oxidized metabolite of aniline. Male Sprague-Dawley rats were given 0.025, 0.05, 0.1, or 0.2 mmol/kg/d of NB in 0.5 ml of 0.25% agar by gavage for 4 d; control rats received the vehicle only. Animals were euthanized at 24 h following the last dose. NB treatment resulted in decreased erythrocyte counts, whereas methemoglobin content increased at 0.1- and 0.2-mmol/kg doses. Spleen weight to body weight ratios were greater by 55 and 81% at O.1- and 0.2-mmol/kg NB doses, respectively. Total iron content in the spleens of NB-treated rats showed dose-dependent significant increases, and the nonheme iron followed a similar pattern. Splenic lipid peroxidation showed a dose-dependent response and was greater by 19, 56, 74, and 85% at the 4 doses, respectively. Malondialdehyde (MDA)-protein adducts, as quantitated by a competitive enzyme-linked immunosorbent assay (ELISA), were markedly greater in all the NB-treated groups, with the highest increase of 248% at 0.2 mmol/kg. Furthermore, NB exposure also resulted in greater protein oxidation (carbonyl content) in the spleens at 0.1- and 0.2-mmol/kg doses. These results suggest that NB is a splenotoxin and therefore can contribute to the splenic toxicity of aniline. Results of this study further support our earlier findings that oxidative stress is a potential mechanism in the splenotoxicity of aniline.

Aniline Compounds↗

PubMed1999

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.

Aniline Compounds↗

PubMed1999

Iron exacerbates aniline-associated splenic toxicity.

Our earlier studies have shown that aniline exposure in rats causes time- and dose-dependent accumulation of iron in the spleen, which may exacerbate aniline splenotoxicity by catalyzing free-radical reactions. The present studies were conducted to test whether aniline-induced splenic toxicity could be potentiated by iron overload. For 30 d male Sprague-Dawley rats received the following treatments: 0.5 mmol/kg/d aniline hydrochloride (AH) by gavage (AH group); 3% carbonyl iron-supplemented diet (IR group); 0.5 mmol/kg/d AH by gavage and iron-supplemented diet (AH + IR group); or no treatments (controls). Treatment-related significant increases in total iron, low molecular weight chelatable iron, lipid peroxidation, and protein oxidation were observed in the spleens of all the groups compared to control. However, these changes were much greater in the combined AH + IR group. The aniline-induced morphological changes in the spleen were consistent with our earlier observations, but were more pronounced in the AH + IR group. The increased toxicity, as evident from greater oxidative stress and morphological changes in the AH + IR group, suggests that iron potentiates the splenic toxicity of aniline.

Aniline Compounds↗

PubMed1999

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.

Anilides↗

PubMed1999

Oxidative modification of lipids and proteins in aniline-induced splenic toxicity.

Our earlier studies with aniline suggested the involvement of oxidative stress as an early toxic event in the spleen. In order to understand the status and consequences of the damaging oxidative reactions, especially during the progression of characteristic splenic lesions, time-dependent subchronic studies were conducted in rats. Male Sprague-Dawley rats were treated with 65 mg/kg/day aniline in the drinking water, while control rats received drinking water only. The animals were euthanized after 1, 2, or 3 months of aniline exposure. Total iron content was remarkably greater in the aniline-treated rats than in age-matched controls. There were time-dependent increases in splenic lipid peroxidation of aniline-treated rats. Malondialdehyde-protein adducts were quantitated by a competitive ELISA and showed greater concentrations in the spleens of aniline-treated rats, further substantiating our lipid peroxidation results. Protein oxidation in the spleens of aniline-treated rats was also greater, with a maximum increase of approximately 76% at 3 months. Western blot analysis for oxidized proteins showed two distinct protein bands at approximately 114 kD and approximately 69 kD in both post-nuclear and mitochondrial fractions of the spleens. Furthermore, densitometric analysis of the blot showed increased band intensities of the oxidized proteins in both these spleen fractions from aniline-treated rats, suggesting the susceptibility of these proteins to aniline-induced oxidative stress. The most prominent morphological changes in the spleens of aniline-treated rats included thickening of the capsule, and capsular cells with nuclear prominence and hyperchromia indicative of capsular hyperplasia. These capsular changes and fibrosis of capsule, splenic trabeculae, and red pulp were noted at all three time points after aniline exposure. Our studies thus suggest that aniline-induced oxidative stress in the spleen is an ongoing event that leads to oxidative modifications of biomolecules. Such oxidative modifications, directly or indirectly, could contribute to the splenic toxicity leading to deleterious consequences, including capsular hyperplasia and fibrosis, as observed in this study, and possibly tumorigenesis in chronic aniline exposure conditions.

Aniline Compounds↗

PubMed1998

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.

Acyltransferases↗

PubMed1998

Phenylhydroxylamine: role in aniline-associated splenic oxidative stress and induction of subendocardial necrosis.

To elucidate the role of N-phenylhydroxylamine (PHA, N-hydroxylated metabolite of aniline) in the selective toxicity of aniline to the spleen, dose-dependent studies were conducted with PHA in rats. Male Sprague-Dawley rats were given four doses each (1 dose/day) of 0.025, 0.05, 0.1, or 0.2 mmol/kg PHA in 0.5 ml of aqueous agar (0.25%) by gavage. The control animals received an equal volume of vehicle only. The animals were euthanized 24 h following the last dose. PHA toxicity in the blood was evident from a dose-dependent increase of methemoglobin. The most affected organ was spleen, which appeared dark and enlarged (splenomegaly) and showed increased spleen-to-body weight ratios, which were 28, 40, 66, and 87% at PHA doses of 0.025, 0.05, 0.1, and 0.2 mmol/kg, respectively. Splenic lipid peroxidation (malondialdehyde content) was higher in all PHA-treated groups, whereas splenic protein oxidation (carbonyl content) increased in only the 0.05, 0.1, and 0.2 mmol/kg groups. The total iron content in the spleen also showed increases of 88, 135, 168, and 209% at PHA doses of 0.025, 0.05, 0.1, and 0.2 mmol/kg, respectively. These biochemical changes were accompanied by a dose-dependent vascular congestion in the spleen, a characteristic feature of aniline toxicity. Although the ratio of organ to body weight increased for both testes and heart at the highest dose, striking morphological changes were observed only in heart. The cardiac lesions consisted of a both acute and resolving multifocal subendocardial necrosis involving predominently the left ventricle. Our results suggest that PHA is a splenotoxin and thus contributes to the toxicity of aniline, while at a high dose, it is also cardiotoxic, perhaps due to anoxia associated with the marked methemoglobinemia. These results further support the involvement of oxidative stress in the splenotoxicity of aniline which may be caused by its reactive metabolite(s) such as PHA.

Aniline Compounds↗

PubMed1998

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.

Animals↗

PubMed1997

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.

Administration, Oral↗

PubMed1997

Oxidative stress in the splenotoxicity of aniline.

Aniline-induced splenic toxicity is characterized by hemorrhage, capsular hyperplasia, fibrosis, and a variety of sarcomas in rats. Early biochemical events responsible for the observed effects are not known. To understand the mechanism(s) of aniline-induced splenic toxicity, single and multiple (four and seven) doses of 1 mmol/kg of aniline hydrochloride(AH) were given in rats. Apart from changes in the hematological parameters, these studies demonstrated that AH could induce lipid peroxidation and protein oxidation in the spleen, and significant increases were observed at four doses. Subsequently, a dose-response study of AH was performed. Male SD rats were given four doses each (one dose/day) of 0.25, 0.5, 1, and 2 mmol/kg of AH in water by gavage, while controls received water only. Animals were euthanized 24 hr following the last dose and tissues obtained. Spleen weight increased by 32 and 80% at 1 and 2 mmol/kg doses, respectively. Splenic lipid peroxidation showed dose-dependent increases of 24, 32, and 43% at 0.5, 1, and 2 mmol/kg, respectively. Protein oxidation in the spleen, quantitated by carbonyl content per milligram protein, showed 10, 28, and 27% increases at 0.5, 1, and 2 mmol/kg, respectively. Iron content in the spleen also showed dose-dependent increases of 72, 172, and 325% at 0.5, 1, and 2 mmol/kg, respectively. Dose-related histopathologic expansion of splenic red pulp was characterized by increasing vascular congestion (most pronounced at 2 mmol/kg), increased red pulp cellularity, erythrophagocytosis, and cellular fragmentation at 1 and 2 mmol/kg; iron deposition in red pulp also increased dramatically with dose. These studies establish that aniline induces lipid peroxidation and protein oxidation in the spleen and suggest that oxidative stress plays a role in the splenic toxicity of aniline.

Administration, Oral↗

PubMed1997

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.

Acyltransferases↗

PubMed1997

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.

Acetates↗