Biomedical subjects

N R Pumford

Publications and source records attributed to N R Pumford.

At least 19 recordsLinked to original sources

PubMed2006

Feed efficiency and mitochondrial function.

Studies have been conducted in our laboratory to assess differences in mitochondrial function and biochemistry in male broilers with high and low feed efficiency (FE) from the same genetic line and fed the same diet. Mitochondria obtained from broilers with low FE exhibited greater uncoupling of the electron transport chain (ETC) that was apparently due to site-specific defects in electron transport resulting in higher amounts of reactive oxygen species (ROS) compared with high FE mitochondria. Higher amounts of ROS production in Low FE mitochondria were likely responsible for higher protein carbonyl levels, indicative of higher protein oxidation compared with High FE mitochondria and tissue. In turn, higher protein damage in Low FE mitochondria may have contributed to lower activity of electron transport chain complexes relative to values observed in high FE mitochondria. Low FE mitochondria did not exhibit a compromised ability to carryout oxidative phosphorylation, and although there were differences in expression of certain electron transport chain proteins, there was nothing that would indicate that differences in coupling and respiratory chain activity could be due to a general decrease in protein expression between low and high FE mitochondria. The results of these studies provide insight into understanding cellular mechanisms associated with the phenotypic expression of feed efficiency in broilers.

Animal Feed↗

PubMed2005

Biochemical evaluation of mitochondrial respiratory chain in duodenum of low and high feed efficient broilers.

Increased H2O2 production, indicating higher oxidative stress, and lower mitochondrial function was previously observed in duodenal mitochondria isolated from broilers with low feed efficiency (FE, gain:feed). Thus, experiments were conducted to 1) evaluate the activity of the respiratory chain complexes (complexes I to V) and 2) assess protein oxidation and mitochondrial protein expression in broilers with low and high FE. Duodenal mitochondria were isolated from broiler breeders with low (0.52 +/- 0.01) and high (0.68 +/- 0.01) FE (n = 8/group). Respiratory chain complex activities were measured spectrophotometrically, whereas mitochondrial protein expression and protein oxidation (carbonyls) were assessed with Western blots. The activities of all complexes, except complex IV, were lower in the low FE compared with high FE mitochondria, whereas protein carbonyl levels were higher in low FE mitochondria. Steady-state levels of 6 out of 7 nuclear-encoded respiratory chain subunits [70S(FP), core I, core II, cytochrome c (cyt c)1, iron-sulfur protein (ISP), and ATPase-alpha] were higher, whereas 3 out of 6 mitochondrial-encoded subunits (ND4, ND6-C, and COX II) were lower in the low FE group, suggesting that sensitivity of mitochondrial proteins to H2O2 or oxidation varies. The general reduction in complex activity and differential protein expression concomitant with higher oxidized proteins in low FE mitochondria suggest that oxidative stress could be contributing to the lower mitochondrial function observed in low FE duodenal mitochondria.

Animal Feed↗

PubMed2005

Compromised liver mitochondrial function and complex activity in low feed efficient broilers are associated with higher oxidative stress and differential protein expression.

Variations in broiler growth and efficiency have been explained in part by differences in mitochondrial function and biochemistry in broilers. To further our knowledge in this regard, 2 experiments were carried out to determine the relationships of a) mitochondrial function and activities of various electron transport chain (ETC) complexes; b) production of H2O2, a reactive oxygen species (ROS), and its association with protein oxidation; and c) mitochondrial protein expression in liver of a single line male broilers with low or high feed efficiency (FE, n = 5 to 8 per group). Mitochondrial function and complex activities were measured polarographically and spectrophotometrically, respectively. H2O2 was measured fluorimetrically, whereas oxidized protein (carbonyls) and specific mitochondrial proteins were analyzed using Western blots. Mitochondrial function (ETC coupling) and activities of ETC complexes (I, II, III, and IV) were higher in high FE compared with low FE broilers. H2O2 and protein carbonyls were higher in the livers of low FE broilers than in high FE broilers. Whereas the expression of 4 immunoreactive proteins [NAD3 (complex I), subunit VII (complex III), cytochrome c oxidase subunits (COX) II, and COX IVb (complex IV)] were higher in low FE liver mitochondria and 2 proteins [subunit 70 (complex II) and a-ATP synthase (complex V)] were higher in high FE birds, there were no differences between groups in the expression of 18 other mitochondrial proteins. In conclusion, increases in oxidative stress in low FE broilers were caused by or may contribute to differences in mitochondrial function (ETC coupling and complex activities) or the differential expression of steady-state levels of some mitochondrial proteins in the liver. Understanding the role of oxidative stress in Low FE broilers will provide clues in understanding the cellular basis of feed efficiency.

Animal Feed↗

PubMed2004

Low feed efficient broilers within a single genetic line exhibit higher oxidative stress and protein expression in breast muscle with lower mitochondrial complex activity.

The objectives of this study were to determine the effects of low or high feed efficiency (FE) on a) protein oxidation, b) the activities of various respiratory chain complexes, and c) expression of various mitochondrial proteins in male broilers within a single genetic line. Tissue homogenate or mitochondria were isolated from breast muscle of broilers with high (0.80 +/- 0.01) and low FE (0.62 +/- 0.02). The complex activities were measured spectrophotometrically, and the levels of oxidized protein (carbonyl) and immunoreactive mitochondrial proteins were analyzed using Western blots. Protein carbonyl levels were higher in low FE compared with high FE broilers breast muscle, which indicated enhanced protein oxidation in low FE mitochondria. Activities of all respiratory chain complexes (I, II, III, IV) were higher in high FE compared with low FE broilers for breast mitochondria. Whereas the expression of immunoreactive proteins was higher in low FE muscle mitochondria for 5 mitochondrial proteins [core I, cyt c1, cyt b (complex III), COX II (cytochrome c oxidase subunit II, complex IV), and adenine nucleotide translocator (ANT1)], there were no differences between groups in the expression of 9 other respiratory chain protein subunits associated with complexex I, II, III, IV, and V. SDS-PAGE revealed a protein band of 47 kDa that was expressed at a higher level in low FE compared with high FE mitochondria. The differential expression of certain mitochondrial proteins and the 47-kDa band might be a compensatory response either to the lower complex activities or increased protein oxidation observed in low FE birds.

Animal Nutritional Physiological Phenomena↗

PubMed2001

Acetaminophen-induced hepatotoxicity in mice lacking inducible nitric oxide synthase activity.

We recently reported that nitrotyrosine and acetaminophen (APAP)-cysteine protein adducts colocalize in the hepatic centrilobular cells following a toxic dose of APAP to mice. Whereas APAP-adducts are formed by reaction of the metabolite N-acetyl-p-benzoquinone imine with cysteine, nitrotyrosine residues are formed by reaction of tyrosine with peroxynitrite. Peroxynitrite is formed from nitric oxide (NO) and superoxide. This manuscript examines APAP (300 mg/kg) hepatotoxicity in mice lacking inducible nitric oxide synthase activity (NOS2 null or knockout mice; C57BL/6-Nos2(tm1Lau)) and in the wildtype mice. In a time course the ALT levels in the exposed NOS2 null mice were approximately 50% of the wildtype mice; however, histological examination of liver sections indicated similar levels of centrilobular hepatic necrosis in both wild-type and NOS2 null mice. Serum nitrate plus nitrite levels (NO synthesis) were identical in saline-treated NOS2 null and wild-type mice (53 +/- 2 microM). APAP increased NO synthesis in wild-type mice only. The increases paralleled the increases in ALT levels with peak levels of serum nitrate plus nitrite at 6 h (168 +/- 27 microM). In wild-type mice hepatic tyrosine nitration was greatly increased relative to saline treated controls. Tyrosine nitration increased in NOS2 null mice also, but the increase was much less. APAP increased hepatic malonaldehyde levels (lipid peroxidation) in NOS2 null mice only. The results suggest the presence of multiple pathways to APAP-mediated hepatic necrosis, one via nitrotyrosine, as in the wild-type mice, and another that is not dependent upon inducible nitric oxide synthase activity, but which may involve increased superoxide.

Acetaminophen↗

PubMed2001

Evaluation of occult acetaminophen hepatotoxicity in hospitalized children receiving acetaminophen. Pediatric Pharmacology Research Unit Network.

The safety of repeated doses of acetaminophen in ill children with the potential of reduced glutathione stores has been questioned. This study measured hepatic transaminases in children and adolescents (n=100) who received > or = 6 therapeutic doses of acetaminophen over a 48-hour period of hospitalization. Acetaminophen-protein adducts were measured in a cohort of subjects with hepatic transaminase elevation (n=8) and in those (n=10) receiving concurrent drug therapy with agents that induce the cytochrome P450 enzymes involved in acetaminophen metabolism. Acetaminophen-protein adducts were not detected in this cohort of 18 subjects. Based on this pilot study, the routine use of acetaminophen at therapeutic doses in ill, hospitalized children and adolescents appears safe.

Acetaminophen↗

PubMed2001

Measurement of acetaminophen-protein adducts in children and adolescents with acetaminophen overdoses. .

Acetaminophen-protein adducts are biomarkers of acetaminophen toxicity present in the centrilobular region of the liver of laboratory animals following the administration of toxic doses of acetaminophen. These biomarkers are highly specific for acetaminophen-induced hepatic injury and correlate with hepatic transaminase elevation. The objective of this prospective, multicenter study was to evaluate the clinical application of the measurement of acetaminophen-protein adducts in pediatric acetaminophen overdose patients. Serum samples were obtained from 51 children and adolescents with acetaminophen overdose at the time of routine blood sampling for clinical monitoring. Six subjects developed "severe" hepatotoxicity (transaminase elevation > 1,000 IU/L), and 6 subjects had transaminase elevation of 100 to 1,000 IU/L. Acetaminophen-protein adducts were detected in the serum of only 1 study subject, a patient with marked transaminase elevation (> 6,000 IU/L) and high risk for the development of hepatotoxicity according to the Rumack nomogram. While this study provides further support for the occurrence of covalent binding of acetaminophen to hepatic protein in humans following acetaminophen overdose, the detection of acetaminophen-protein adducts in serum with the current methodology requires significant biochemical evidence of hepatocellular injury.

Acetaminophen↗

PubMed2000

Trichloroethylene accelerates an autoimmune response by Th1 T cell activation in MRL +/+ mice.

Trichloroethylene (1,1,2-trichloroethene) is a major environmental contaminant. There is increasing evidence relating exposure to trichloroethylene with autoimmunity. To investigate potential mechanisms, we treated the autoimmune-prone MRL +/+ mice with trichloroethylene in the drinking water at 0, 2.5 or 5.0 mg/ml and sacrificed them at 4, 8 and 22 weeks. As early as 4 weeks of treatment, Western blot analysis showed a dose-dependent increase in the level of trichloroethylene-modified proteins, indicating that a reactive metabolite of trichloroethylene was formed. Significant increases in antinuclear antibodies (ANA) and total serum immunoglobulins were found following 4-8 weeks of trichloroethylene treatment, indicating that trichloroethylene was accelerating an autoimmune response. Investigation into possible mechanisms of this autoimmune response revealed that trichloroethylene treatment dramatically increased the expression of the activation marker CD44 on splenic CD4+ T cells at 4 weeks. In addition, splenic T cells from mice treated for 4 weeks with trichloroethylene secreted more IFN-gamma and less IL-4 than control T cells, consistent of a T-helper type 1 (Th1) type immune or inflammatory response. A specific immune response directed against dichloroacetylated proteins was found at 22 weeks of trichloroethylene treatment. Taken collectively, the results suggest that trichloroethylene treatment accelerated an autoimmune response characteristic of MRL +/+ mice in association with nonspecific activation of Th1 cells. In addition, long-term treatment with trichloroethylene led to the initiation of a trichloroethylene-specific immune response.

Animals↗

PubMed2000

Western blot analysis for nitrotyrosine protein adducts in livers of saline-treated and acetaminophen-treated mice.

The hepatic centrilobular necrosis produced by the analgesic/antipyretic acetaminophen correlates with metabolic activation of the drug leading to its covalent binding to protein. However, the molecular mechanism of the toxicity is not known. Recent immunohistochemical analyses using an antinitrotyrosine antiserum indicated that nitrotyrosine protein adducts co-localized with the acetaminophen-protein adducts in the centrilobular cells of the liver. Nitration of proteins is believed to occur by peroxynitrite, a substance formed by the rapid reaction of superoxide with nitric oxide. Nitric oxide and superoxide may be formed by activated Kupffer cells or by other cells. Because we were unable to successfully utilize the commercial antiserum in Western blot analyses of liver fractions, we developed a new antiserum. With our antiserum, liver fractions from saline-treated control and acetaminophen-treated mice were successfully analyzed for nitrated proteins. The immunogen for this new antiserum was synthesized by coupling 3-nitro-4-hydroxybenzoic acid to keyhole limpet hemocyanin. A rabbit immunized with this adduct yielded a high titer of an antiserum that recognized BSA nitrated with peroxynitrite. Immunoblot analysis of nitrated BSA indicated that nitrotyrosine present in a protein sample could be easily detected at levels of 20 pmoles. Immunohistochemical analyses indicated that nitrotyrosine protein adducts were detectable in the centrilobular areas of the liver. Immunoblot analysis of liver homogenates from both saline-treated and acetaminophen-treated mice (300 mg/kg) indicate that the major nitrotyrosine protein adducts produced have molecular weights of 36 kDa, 44 kDa, and 85 kDa. The 85-kDa protein stained with the most intensity. The hepatic homogenates of the acetaminophen- treated mice showed significantly increased levels of all protein adducts.

Acetaminophen↗

PubMed2000

Inhibition of CYP2E1 reverses CD4+ T-cell alterations in trichloroethylene-treated MRL+/+ mice.

Trichloroethylene is an organic solvent that is primarily used as a degreasing agent for metals. There is increasing evidence in both humans and animal models that trichloroethylene promotes the development of autoimmunity, but little is known about the mechanisms that mediate the effect of trichloroethylene on the immune system. Metabolic activation of trichloroethylene is considered an obligatory pathway for other known toxicities such as hepatotoxicity, nephrotoxicity, and carcinogenicity. Trichloroethylene is metabolized by the cytochromes P450, primarily cytochrome P450 2E1 (CYP2E1). To investigate whether metabolism by CYP2E1 is required for immunomodulation, we treated autoimmune prone MRL+/+ mice with trichloroethylene in the drinking water for 4 weeks, in the presence or absence of diallyl sulfide, a specific inhibitor of CYP2E1. Using an antibody that recognizes proteins covalently modified by a reactive metabolite of trichloroethylene; two immunoreactive proteins were detected in liver microsomes from trichloroethylene-treated mice. Formation of these trichloroethylene-protein adducts, an indicator of metabolic activation, was completely inhibited in animals that were concomitantly treated with trichloroethylene and diallyl sulfide. The level of CYP2E1 apoprotein in liver microsomes was significantly reduced in the presence of diallyl sulfide. The enhanced mitogen-induced proliferative capacity of T cells from trichloroethylene-treated MRL+/+ mice was inhibited if the mice were also treated with diallyl sulfide. In addition, the reduction in interleukin-4 levels secreted by activated CD4+ T cells from trichloroethylene-treated mice was reversed if the mice were also treated with diallyl sulfide. Taken collectively, metabolism of trichloroethylene by CYP2E1 is responsible, at least in part, for the CD4+ T cell alterations associated with exposure to this environmental toxicant.

Allyl Compounds↗

PubMed2000

CD4(+) T-cell activation and induction of autoimmune hepatitis following trichloroethylene treatment in MRL+/+ mice.

Exposure to relatively high levels of trichloroethylene has recently been shown to accelerate the development of an autoimmune response in the autoimmune prone MRL+/+ mice. The trichloroethylene-induced autoimmune response was associated with an increase in activated CD4(+) T cells, producing Th(1)-like cytokines. The present study was conducted to determine whether lower, more occupationally relevant doses of trichloroethylene could also promote autoimmunity, in MRL+/+ mice, and if so, to investigate the mechanism of this accelerated autoimmune response. In addition, histological studies were performed to determine if trichloroethylene was capable of producing pathological markers consistent with an autoimmune disease. Trichloroethylene was administered to mice in the drinking water at 0, 0.1, 0.5, and 2.5 mg/ml for 4 and 32 weeks. There was a significant increase above controls in serum antinuclear antibody (ANA) levels following 4 weeks of both 0.1 and 0.5 mg/kg/day of trichloroethylene. After 32 weeks of treatment, ANA levels were elevated and equal in all groups. The kinetics of the ANA response indicated that trichloroethylene accelerated the innate autoimmune response in the MRL+/+ mice. There was a dose-related increase in the percentage of activated CD4(+) T cells in both the spleens and lymph nodes of mice treated for 32 weeks with trichloroethylene when compared to controls. CD4(+) T cells isolated from MRL+/+ mice after either 4 or 32 weeks of treatment with trichloroethylene secreted inflammatory or Th(1)-like cytokines. Following 32 weeks of trichloroethylene treatment, there was a significant increase in hepatic mononuclear infiltration localized to the portal region, a type of hepatic infiltration consistent with autoimmune hepatitis. Taken collectively, these data suggest that exposure to occupationally relevant concentrations of trichloroethylene can accelerate an autoimmune response and can lead to autoimmune disease. The mechanism of this autoimmunity appears to involve, at least in part, activated CD4(+) T cells that then produced inflammatory cytokines.

Animals↗

PubMed1999

Deferoxamine delays the development of the hepatotoxicity of acetaminophen in mice.

The hepatotoxicity of acetaminophen is conventionally ascribed to metabolism by CYP450 to N-acetyl-p-benzoquinone imine and covalent binding to proteins. We investigated a potential role for oxidative stress by determining the effect of the ferric chelator deferoxamine (Desferal) on acetaminophen (paracetamol)-induced hepatotoxicity in mice. Administration of deferoxamine (75 mg/kg) 1 h after a toxic dose of acetaminophen (300 mg/kg) significantly delayed the development of the toxicity without altering covalent binding. In saline-treated mice serum ALT was 18 +/- 2 IU/l. In acetaminophen-treated mice serum alanine aminotransferase (ALT) was 779 +/- 271 at 2 h, 7421 +/- 552 IU/l at 4 h, 5732 +/- 523 IU/l at 8 h, and 5984 +/- 497 IU/l at 24 h. In acetaminophen plus deferoxamine-treated mice, serum ALT was 80 +/- 10 at 2 h, 472 +/- 74 IU/l at 4 h, 2149 +/- 597 IU/l at 8 h, and 5766 +/- 388 at 24 h. Deferoxamine at 1 h after acetaminophen did not decrease serum ALT at 12 h; however, deferoxamine at 1 and 4 h, or deferoxamine at 1 h plus N-acetylcysteine at 4 h to replete hepatic glutathione, decreased the toxicity from 5625 +/- 310 IU/l to 3436 +/- 546 IU/l and 3003 +/- 282 IU/l, respectively. Deferoxamine plus N-acetylcysteine at 1.25 h after acetaminophen was more effective at decreasing the 24 h toxicity than N-acetylcysteine alone. In acetaminophen treated mice, higher doses of deferoxamine (150-300 mg/kg) at 1 h greatly increased the observed hepatotoxicity at 4 h in a dose responsive manner, but deferoxamine alone was nontoxic.

Acetaminophen↗

PubMed1999

Pretreatment of mice with macrophage inactivators decreases acetaminophen hepatotoxicity and the formation of reactive oxygen and nitrogen species.

Hepatotoxic doses of acetaminophen to mice produce not only acetaminophen-protein adducts in the centrilobular cells of the liver, but nitrotyrosine-protein adducts in the same cells, the site of the necrosis. Nitration of tyrosine occurs with peroxynitrite, a species formed by reaction of nitric oxide (NO.) with superoxide (O2. -). Because NO. and O2.- may be produced by activated Kupffer cells and/or infiltrated macrophages, we pretreated mice with the macrophage inactivators/depeleters gadolinium chloride (7 mg/kg, intravenously [iv]) or dextran sulfate (10 mg/kg, iv) 24 hours before administration of acetaminophen (300 mg/kg). Mice treated with acetaminophen plus gadolinium chloride, or acetaminophen plus dextran sulfate, had significantly less evidence of hepatotoxicity as evidenced by lower serum alanine transaminase (ALT) levels (28 +/- 1 IU/L and 770 +/- 240 IU/L, respectively) at 8 hours compared with acetaminophen (6,380 +/- 408 IU/L). Analysis of hepatic homogenates for acetaminophen-protein adducts at 2 hours, a time of maximal covalent binding and before hepatocyte lysis, indicated that these pretreatments did not decrease covalent binding. Western blot analysis for the macrophage marker protein F4/80 in homogenates revealed not only the expected decrease by the macrophage inactivators/depleters, but also an apparent increase in acetaminophen-only-treated mice. At 8 hours nitrotyrosine-protein adducts were present in the acetaminophen-only-treated mice, but not in the acetaminophen plus gadolinium chloride-treated mice, or acetaminophen plus dextran sulfate-treated mice. High levels of heme-protein adducts, a measure of oxidative stress, were detected in livers of the 8 hour acetaminophen-only-treated mice. These data suggest that acetaminophen hepatotoxicity is mediated by an initial metabolic activation and covalent binding, and subsequent activation of macrophages to form O2.-, NO., and peroxynitrite. Nitration of tyrosine correlates with toxicity.

Acetaminophen↗

PubMed1999

Trichloroethylene activates CD4+ T cells: potential role in an autoimmune response.

Trichloroethylene is an industrial solvent and has become a major environmental contaminant. Autoimmune-prone MRL +/+ mice were treated for up to 22 weeks with trichloroethylene in the drinking water (0, 2.5, and 5.0 mg/mL) in order to study the immunoregulatory effects of this environmental toxicant. After only 4 weeks of treatment, trichloroethylene was shown to promote the expansion of CD4+ T cells that expressed a memory/activation phenotype (i.e., CD44hi CD45RBlo) and secreted high levels of IFN-gamma, but not IL-4. In addition, trichloroethylene treatment accelerated the development of an autoimmune response in the MRL +/+ mice as evidenced by an earlier appearance of antinuclear antibodies and increased levels of total IgG2a. MRL +/+ mice treated with trichloroethylene for 22 weeks also contained antibodies specific for trichloroethylene adducts, suggesting the activation of trichloroethylene-specific T cells. The results suggest that trichloroethylene can stimulate antigen nonspecific as well as specific T cells that are capable of promoting autoimmunity in genetically predisposed individuals.

Animals↗

PubMed1998

The acetaminophen regioisomer 3'-hydroxyacetanilide inhibits and covalently binds to cytochrome P450 2E1.

3'-Hydroxyacetanilide has been previously studied as a nontoxic regioisomer of the analgesic acetaminophen (4'-hydroxyacetanilide). The radiolabeled derivative has been shown to covalently bind to liver proteins at levels similar to that observed with hepatotoxic doses of radiolabeled acetaminophen with no evidence of hepatic damage. Using an anti-arylacetamide antiserum the primary protein adduct detected following administration of 3'-hydroxyacetanilide (300 and 600 mg/kg) to mice was a 50 kDa microsomal protein that co-migrated with cytochrome P450 2E1. Cytochrome P450 2E1 enzyme activity (p-nitrophenol hydroxylase) was decreased by 79% in the mice treated with 3'-hydroxyacetanilide (600 mg/kg). Incubation of 3'-hydroxyacetanilide with hepatic microsomes resulted in a time dependent 47% decrease in cytochrome P450 2E1 activity. Pre-incubation of acetaminophen with microsomes did not result in covalent binding to the cytochrome P450 nor was there a decrease in p-nitrophenol hydroxylase activity. These data suggest that 3'-hydroxyacetanilide covalently binds to cytochrome P450 2E1 with preferential loss of activity.

Acetaminophen↗

PubMed1998

Covalent binding of trichloroethylene to proteins in human and rat hepatocytes.

The environmental contaminant and occupational solvent trichloroethylene is metabolized to a reactive intermediate that covalently binds to specific hepatic proteins in exposed mice and rats. In order to compare covalent binding between humans and rodents, primary hepatocyte cultures were exposed to vaporized trichloroethylene at 0-10,000 parts per million for up to 2 h. Immunochemical detection of three major dose- and time-dependent trichloroethylene protein adducts at 50, 52 and 100 kDa was demonstrated in the rat hepatocytes, while a single, distinctively different 47 kDa adduct was detected in human hepatocytes. The 50 kDa adduct in rat hepatocytes was found to comigrate on SDS-PAGE with cytochrome P450 2E1 (CYP2E1), while the adduct found in humans did not comigrate with CYP2E1. These data show that reactive metabolites of trichloroethylene can be formed in human and rat hepatocytes and bind covalently to discrete hepatic proteins, and suggests that in rats, but not humans, that one of the targets is CYP2E1.

Animals↗

PubMed1998

Nitrotyrosine-protein adducts in hepatic centrilobular areas following toxic doses of acetaminophen in mice.

Treatment of mice with a toxic dose of acetaminophen (300 mg/kg, ip) significantly increased hepatotoxicity at 4 h, as evidenced by histological necrosis in the centrilobular areas of the liver, and increased serum levels of alanine aminotransferase (ALT) (from 8 +/- 1 IU/L in saline-treated mice to 3226 +/- 892 IU/L in the acetaminophen-treated mice). Serum levels of nitrate plus nitrite (a marker of nitric oxide synthesis) were also increased from 62 +/- 8 microM in saline-treated mice to 110 +/- 14 microM in acetaminophen-treated mice (P < 0.05). Regression analysis of serum ALT levels to serum nitrate plus nitrite levels in individual mice revealed a positive, linear relationship between serum ALT levels and serum nitrate plus nitrite levels with a correlation coefficient of 0.9 (P < 0.05). The y intercept value (nitrate plus nitrite level) was 63 +/- 15 microM. Immunohistochemical analysis of liver sections from acetaminophen-intoxicated mice using an anti-3-nitrotyrosine antibody indicated tyrosine nitration in the proteins of the centrilobular cells. Tyrosine nitration has been shown to occur by peroxynitrite, a reactive intermediate formed by an extremely rapid reaction of nitric oxide and superoxide and a species which also has hydroxyl radical-like activity. Analysis of liver sections using an anti-acetaminophen antiserum indicated the centrilobular cells also contained acetaminophen-protein adducts, a reaction of the metabolite N-acetyl-p-benzoquinone imine with cysteine residues on proteins. These data are consistent with acetaminophen metabolic activation leading to increased synthesis of nitric oxide and superoxide and to peroxynitrite as an important intermediate in the toxicity.

Acetaminophen↗

PubMed1998

Immunochemical detection of quinol--thioether-derived protein adducts.

Glutathione (GSH) conjugates of hydroquinone (HQ) and 2-bromohydroquinone (2-BrHQ) produce severe renal proximal tubular necrosis in rats. Since the reactivity of quinones lies, in part, in their ability to alkylate proteins, our goal was to develop an immunochemical method with which to investigate the role of protein adduct formation in quinone-thioether-mediated toxicity. An immunogen was synthesized by coupling 2-bromo-6-(N-acetylcystein-S-yl)hydroquinone (2-BrHQ-NAC) to keyhole-limpet hemocyanin (KLH). Anti-2-BrHQ-NAC-KLH antibodies were raised in rabbits and purified by affinity chromatography. Antibody binding to the 2-BrHQ-NAC epitope was confirmed by competitive enzyme-linked immunosorbent assay (ELISA) with a bovine serum albumin conjugate of 2-BrHQ-NAC. Affinity-purified anti-2-BrHQ-NAC-KLH antibodies recognized adducted proteins in the kidneys of rats treated with HQ, 2-BrHQ, 2-bromo-bis(glutathion-S-yl)hydroquinone, 2-(glutathion-S-yl)hydroquinone, 2, 5-bis(glutathion-S-yl)hydroquinone, and 2,3, 5-tris(glutathion-S-yl)hydroquinone. Immunoreactive proteins were found in all renal subcellular fractions of 2-BrHQ-treated rats, and the distribution of adducts was similiar to that obtained by quantifying 2-Br[14C]HQ covalent adducts. Western blot analysis revealed that three proteins, at 42, 46, and 79 kDa, were adducted by all the compounds examined. The identification of these adducted proteins will be required to assess their significance in quinol-thioether-mediated nephrotoxicity.

Animals↗