Biomedical subjects

H Raza

Publications and source records attributed to H Raza.

At least 19 recordsLinked to original sources

PubMed2005

Safety profile of the intravitreal streptokinase-plasmin complex as an adjunct to vitrectomy in the rabbit.

BACKGROUND: The generation of an atraumatic posterior vitreous detachment (PVD), a common goal in vitreoretinal surgery, is a challenge, particularly in children and young trauma patients. Plasmin has been proposed as an adjunct to vitrectomy to enzymatically generate a PVD. Low doses of streptokinase-activated plasmin were tested in human pilot studies. This dose-escalation study assesses the safety range of intravitreal human streptokinase-plasmin in rabbits. METHODS: Plasminogen was isolated from human plasma by affinity chromatography, followed by activation with streptokinase (1:1), to generate the streptokinase-plasmin complex. Enzyme doses from 0.1-7 activity units (AU, in 0.1 ml) were injected into the mid-vitreous of 35 eyes; six control eyes were injected with balanced salt solution (BSS, 0.1 ml). Thirty minutes after injection, a two-port vitrectomy was performed. Fundus and slit lamp examinations were performed on days 1 and 7. On days 2 and 7, bright flash electroretinography was performed and compared with preoperative recordings. Some animals receiving higher doses of streptokinase-plasmin (1-7 AU) were followed clinically and with electroretinography for up to 9 months. RESULTS: A mild-to-moderate inflammatory response was seen in both control and plasmin-treated eyes on day 1, but had disappeared completely by day 7 in most eyes. In the 7 AU group, inflammation was stronger and more protracted. Two of three eyes from this group developed wrinkling of the medullary rays; one of them showed discoloration and traction at the medullary rays in the late follow-up. Electroretinograms (ERGs) of vitrectomized control eyes showed the following changes from preoperative values: 48 h, a-wave -11.10% [no significant (n.s.)], b-wave -14.62% (P=0.046); 7 days, a-wave +9.18% (n.s.), b wave +11.69% (n.s.). For the enzyme-treated eyes: 48 h: a-wave -20.43% (P<0.001), b-wave -9.57% (p<0.001); 7 days: a wave -14.21% (P<0.001), b-wave +2.48% (P<0.001). There was no evidence of dose-dependent ERG changes in enzyme-treated eyes at doses up to 5 AU. Groups of up to 3 AU were investigated by light and transmission electron microscopy, without evidence of toxicity. CONCLUSION: Streptokinase-plasmin doses up to 3 AU were found to be safe when injected into rabbit eyes followed by vitrectomy.

Animals↗

PubMed2005

Can renal scan findings predict biopsy-proven allograft rejection?

OBJECTIVES: To assess the usefulness of isotopic renogram in diagnosing acute renal graft rejection. MATERIALS AND METHODS: Degree of perfusion and allograft uptake of tracer were correlated with the clinical and biopsy diagnoses in 15 postrenal transplant patients with varying degrees of renal impairment. Renographic findings and perfusion calculations were done by a blinded observer. RESULTS: A strong correlation was found between renal histology and renal scan findings in 13 of 15 patients. Sensitivity and specificity of renal scanning in diagnosing acute rejection were 85% and 50% respectively (using renal biopsy findings as the gold standard). CONCLUSION: Our results demonstrate a strong correlation between blinded perfusion assessment and biopsy-proven acute rejection. We conclude, therefore, that single renal flow scan with DTPA (noninvasive/nonnephrotoxic) allows a physician to tailor therapy for acute renal graft dysfunction. We suggest that in cases with a renographic diagnosis of AR, the patient should receive standard antirejection therapy. Renal biopsy should be reserved for those instances when the renographic findings are not definitive and those when the patient fails to respond to a standard methylprednisolone therapy.

Biopsy↗

PubMed2002

Differential modulation of growth and glutathione metabolism in cultured rat astrocytes by 4-hydroxynonenal and green tea polyphenol, epigallocatechin-3-gallate.

Oxidative stress has been implicated in the pathogenesis of cancer and prominent neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. Apoptosis and cell cycle deregulation appear to be the mode of cell death in these disorders. Green tea polyphenol, epigallocatechin-3-gallate (EGCG) has been shown to be a potent antiinflammatory, apoptotic and cancer chemopreventive agent. 4-Hydroxynonenal (HNE), a by-product of lipid peroxidation (LPO), has been reported to induce apoptosis and inhibit growth in many cell systems including neuroglial cultures. We have studied both the dose and time dependent effects of HNE and EGCG on the viability of primary astrocyte cell cultures prepared from neonatal rats. HNE was found to be cytotoxic at a higher dose (0.1 mM) and markedly reduced (up to 80%) the astrocyte viability while EGCG did not appear to be cytotoxic under similar conditions. In addition, we have also studied the alterations in glutathione (GSH) and LPO levels and the activities of GSH metabolizing enzymes after treatment with HNE and EGCG. A 40% decrease in GSH level and a moderate increase in LPO were observed in HNE treated cells suggesting an increase in oxidative stress. HNE treatment caused a 50% decrease in GSH reductase and a 35% increase in GSH peroxidase activities. Although HNE treatment did not lead to any significant alterations in GSH-S-transferase (GST) activity, an increased expression of GST isoenzymes was seen following the exposure to HNE. EGCG treatment caused a significant increase in LPO even in the presence of elevated GSH content. In contrast to HNE, EGCG treatment resulted in a significant decrease (50%) in the activity and expression of GSTs. Treatment of astrocyte cultures with HNE, resulted in a severe impairment in mitochondrial respiration as measured by MTT exclusion assay, while treatment with EGCG had no effect on mitochondrial respiratory activity. Both HNE and EGCG were found to initiate apoptosis in astrocytes as measured by DNA fragmentation assay. However, HNE seems to be a stronger apoptotic and cytotoxic agent than EGCG. These results suggest that HNE and EGCG differentially modulate oxidative stress and regulate the growth and survival of astrocytes.

Aldehydes↗

PubMed2001

Hypotriglyceridemic and hypocholesterolemic effects of anti-diabetic Momordica charantia (karela) fruit extract in streptozotocin-induced diabetic rats.

Momordica charantia (karela) is commonly used as an antidiabetic and antihyperglycemic agent in Asian, Oriental and Latin American countries. This study was undertaken to investigate the effects of long term feeding (10 weeks) of M. charantia fruit extract on blood plasma and tissue lipid profiles in normal and streptozotocin (STZ)-induced Type 1 diabetic rats. The results show that there was a significant (P < 0.05) increase in plasma non-esterified cholesterol, triglycerides and phospholipids in STZ-induced diabetic rats, accompanied by a decrease in high density lipoprotein (HDL)-cholesterol. A moderate increase in plasma (LPO) product, malonedialdehyde (MDA), and about two-fold increase in kidney LPO was also observed in STZ-induced diabetic rats. The treatment of diabetic rats with M. charantia fruit extract over a 10-week period returned these levels close to normal. In addition, karela juice also exhibited an inhibitory effect on membrane LPO under in vitro conditions. These results suggest that M. charantia fruit extract exhibits hypolipidemic as well as hypoglycemic effects in the STZ-induced diabetic rat.

Animals↗

PubMed2000

Modulation of xenobiotic metabolism and oxidative stress in chronic streptozotocin-induced diabetic rats fed with Momordica charantia fruit extract.

We studied the long-term effects of streptozotocin-induced diabetes on tissue-specific cytochrome P450 (CYP) and glutathione-dependent (GSH-dependent) xenobiotic metabolism in rats. In addition, we also studied the effect of antidiabetic Momordica charantia (karela) fruit-extract feeding on the modulation of xenobiotic metabolism and oxidative stress in rats with diabetes. Our results have indicated an increase (35-50%) in CYP4A-dependent lauric acid hydroxylation in liver, kidney, and brain of diabetic rats. About a two-fold increase in CYP2E-dependent hepatic aniline hydroxylation and a 90-100% increase in CYP1A-dependent ethoxycoumarin-O-deethylase activities in kidney and brain were also observed. A significant increase (80%) in aminopyrene N-demethylase activity was observed only in rat kidney, and a decrease was observed in the liver and brain of diabetic rats. A significant increase (77%) in NADPH-dependent lipid peroxidation (LPO) in kidney of diabetic rats was also observed. On the other hand, a decrease in hepatic LPO was seen during chronic diabetes. During diabetes an increased expression of CYP1A1, CYP2E1, and CYP4A1 isoenzymes was also seen by Western blot analysis. Karela-juice feeding modulates the enzyme expression and catalytic activities in a tissue- and isoenzyme-specific manner. A marked decrease (65%) in hepatic GSH content and glutathione S-transferase (GST) activity and an increase (about two-fold) in brain GSH and GST activity was observed in diabetic rats. On the other hand, renal GST was markedly reduced, and GSH content was moderately higher than that of control rats. Western blot analyses using specific antibodies have confirmed the tissue-specific alterations in the expression of GST isoenzymes. Karela-juice feeding, in general, reversed the effect of chronic diabetes on the modulation of both P450-dependent monooxygenase activities and GSH-dependent oxidative stress related LPO and GST activities. These results have suggested that the modulation of xenobiotic metabolism and oxidative stress in various tissues may be related to altered metabolism of endogenous substrates and hormonal status during diabetes. The findings may have significant implications in elucidating the therapeutic use of antidiabetic drugs and management of Type 1 diabetes in chronic diabetic patients.

Animals↗

PubMed2000

Cyclic adenosine monophosphate regulates the expression of the intercellular adhesion molecule and the inducible nitric oxide synthase in brain endothelial cells.

The authors studied whether cyclic AMP (cAMP), a widespread regulator of inflammation, modulates the cytokine-mediated expression of the intercellular adhesion molecule, intercellular adhesion molecule-1 (ICAM-1), and the inflammatory nitric oxide synthase 2 (NOS-2), in primary and immortalized brain endothelial cell cultures (GP8.3 cell line). When measured by enzyme-linked immunosorbent assay (ELISA), ICAM-1 was constitutively expressed and was up-regulated twofold by interleukin-1beta, with no effect of interferon-gamma. The NOS-2 activity, assessed by nitrite accumulation, was absent from untreated cultures but was induced by interleukin-1beta and interferon-gamma acting synergistically. Stimulation of cAMP-dependent pathways with forskolin or dibutyryl cAMP decreased ICAM-1 protein expression, whereas it increased NOS-2 protein expression. For both ICAM-1 and NOS-2, mRNA expression correlated with protein expression. Blockade of NOS activity with L-N-monomethylargiuine (L-NMMA) did not alter ICAM-1 expression, indicating that the nitric oxide released by NOS-2 did not cause the down-regulation of ICAM-1. Analysis of NFKB activation indicated that cAMP acted through a mechanism other than inhibition of nuclear translocation of NFKB. The authors conclude that cAMP modulates the expression of proinflammatory molecules in brain endothelium. This suggests that inflammatory processes at the blood-brain barrier in vivo may be regulated by perivascular neurotransmitters via cAMP.

Animals↗

PubMed2000

Proto-oncogene ras GTPase-linked induction of glutathione-S-transferase by growth factors in PC12 cells.

This report provides evidence linking activation of Ras GTPase by growth factors and induction of glutathione-S-transferase isozymes in PC12 cells. Ras GTPase was activated by EGF, NGF, insulin and phorbolester in PC12 cells. Activation of Ras GTPase was found to be associated with induction of the expression of GST mu and pi isoenzymes while there was no detectable induction of GST alpha expression. GST pi was found to be induced by all the Ras GTPase activating agents tested while activation of Ras by phorbolester and insulin induced expression of GST mu only. These results suggest a role of Ras, at least in part, in controlling the expression of GST and that there might be independent signalling pathways for the expression of different GST isoenzymes. GST activity was found to be very high (4-fold) in the lysate obtained from retinoic acid treated PC12 cells when compared with untreated cells. Induction of GST expression was found to be initiated within 30 min of retinoic acid treatment in PC12 cells reaching a maximum level at 4 h. However, immunoblot analysis showed that retinoic acid (RA), unlike mitogens/growth factors, weakly induced the expression of GST pi but not the expression of alpha, mu and microsomal GSTs. Overxpression of inhibitory polypeptides that block signals generated from Ras and Cdc42 was found to reverse the retinoic acid activation-dependent induction of GST expression in PC12 cells. These results provide evidence for the first time suggesting a novel role of Ras GTPase in the regulation of GST expression which might have a significant implication in developing drug resistance and/or growth of cancer cells.

Animals↗

PubMed1999

Constitutive and inducible cytochromes P450 in rat lung mitochondria: xenobiotic induction, relative abundance, and catalytic properties.

The presence of xenobiotic-inducible CYP1A1, 2B1/2, and 3A1/2 in rat lung mitochondria was investigated using mitochondrial preparations of defined purity. The mitochondrial P450 content in uninduced lung was 1.5-fold higher compared to microsomes. Administration of BNF induced the P450 contents by twofold in both mitochondrial and microsomal membrane fractions. BNF treatment induced EROD activity to about 40-fold in the microsomal fraction and 25-fold in the mitochondrial fraction. The microsomal induction was observed at 4 days of BNF treatment, while the mitochondrial induction required 10 days of treatment. Consistent with the activity profile, Western blot analysis showed the presence of CYP1A1 antibody reactive protein only in lung mitochondria from BNF-treated rats. BNF administration also caused a 50 to 80% reduction in the CYP2B1/2-associated PROD and BROD activities and CYP3A1/2-associated ERND activity in both mitochondria and microsomes. There was also a parallel reduction in the antibody reactive CYP2B1/2 and 3A1/2 proteins in both of these membrane fractions. Administration of DEX for 4 days induced mitochondrial and microsomal ERND activity by 1. 7- and 2.5-fold, respectively. Mitochondrial EROD activity was inhibited by antibodies to P450MT2, as well as Adx, but not by antibody against P450 reductase, indicating the mitochondrial localization of CYP1A1. Protease protection and alkaline extraction experiments indicated that CYP1A1 associated with lung mitochondria is localized inside the inner membrane and exists as a membrane extrinsic protein. In summary, this is probably the first report of inducible P450s in rat lung mitochondria, and our results suggest a possible functional role for these mitochondrial enzymes in xenobiotic metabolism.

Animals↗

PubMed1998

Preferential effects of nicotine and 4-(N-methyl-N-nitrosamine)-1-(3-pyridyl)-1-butanone on mitochondrial glutathione S-transferase A4-4 induction and increased oxidative stress in the rat brain.

We have investigated the in vivo effects of the tobacco-specific toxins nicotine and 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) on antioxidant defense systems in the mitochondrial, microsomal, and cytosolic compartments of rat brain, lung, and liver. Nicotine induced maximum oxidative stress in brain mitochondria, as seen from a 1.9-fold (P < 0.001) increase in thiobarbituric acid-reactive substance (TBARS) and a 2-fold (P < 0.001) increase in glutathione S-transferase (GST) A4-4 (also referred to as rGST 8-8) activities. These changes were accompanied by a 25-40% increase in reactive oxygen species and a 20-30% decrease in alcohol dehydrogenase activities. The 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone-induced oxidative damage was apparent in the microsomal fraction of brain, lung, and liver, and it also increased 4-hydroxynonenal specific GST A4-4 activity in the brain and lung mitochondrial matrix fraction. The levels of microsomal thiobarbituric acid reactive substance, cytochrome P4502E1 activity, and reactive oxygen species were also increased significantly (P < 0.001) in all tissues. Both of these toxins induced the level of GST A4-4 mRNA in the brain, while they caused a marked reduction in the liver GST A4-4 mRNA pool. Additionally, the brain mitochondrial matrix showed a markedly higher level of 4-hydroxynonenal specific GST activity and mGST A4-4 antibody-reactive protein than did the cytosolic fraction. In conclusion, the present study provides evidence for the occurrence of GST A4-4 enzyme activity in mammalian mitochondria, in addition to demonstrating that both mitochondria and microsomes are intracellular targets for nicotine- and NNK-induced organ toxicity.

Animals↗

PubMed1998

Drug metabolizing enzyme systems in the houbara bustard (Chlamydotis undulata).

This study compared catalytic and immunochemical properties of drug metabolizing phase I and II enzyme systems in houbara bustard (Chlamydotis undulata) liver and kidney and rat liver. P450 content in bustard liver (0.34 +/- 0.03 nmol mg-1 protein) was 50% lower than that of rat liver (0.70 +/- 0.02 nmol mg-1 protein). With the exception of aniline hydroxylase activity, monooxygenase activities using aminopyrine, ethoxyresorufin and ethoxycoumarin as substrates were all significantly lower than corresponding rat liver enzymes. As found in mammalian systems the P450 activities in the bird liver were higher than in the kidney. Immunohistochemical analysis of microsomes using antibodies to rat hepatic P450 demonstrated that bustard liver and kidney express P4502C11 homologous protein; no appreciable cross-reactivity was observed in bustards using antibodies to P4502E1, 1A1 or 1A2 isoenzymes. Glutathione content and glutathione S-transferase (GST) activity in bustard liver were comparable with those of rat liver. GST activity in the kidney was 65% lower than the liver. Western blotting of liver and kidney cytosol with human GST isoenzyme-specific antibodies revealed that the expression of alpha-class of antibodies exceeds mu in the bustard. In contrast, the pi-class of GST was not detected in the bustard liver. This data demonstrates that hepatic and renal microsomes from the bustard have multiple forms of phase I and phase II enzymes. The multiplicity and tissue specific expression of xenobiotic metabolizing enzymes in bustards may play a significant role in determining the pharmacokinetics of drugs and susceptibility of the birds to various environmental pollutants and toxic insults.

Animals↗

PubMed1998

Multiplicity and tissue specific expression of camel cytochrome P450(s).

The presence of multiple forms of the cytochrome P450 was demonstrated enzymatically in camel tissues using a variety of isoenzyme specific substrates and immunochemically using isoenzyme specific antibodies. The maximum catalytic activity using xenobiotics as substrate was observed in the liver followed by the kidney. However, lauric acid hydroxylation was found to be higher in the kidney than in any other tissues. Camel liver microsomal monooxygenase activity using aniline, aminopyrene, ethoxycoumarin, ethoxyresorufin and benzo(a)pyrene as substrates was comparable with those of rat and human livers. The activity of the enzymes in extrahepatic tissues of the camel was comparable with those of the rat extrahepatic tissues. The maximum expression of P450 protein was seen in the camel liver and kidney while the brain and intestine exhibited relatively low levels of expression. P450 expression in camel tissues appeared to be higher than in rat tissues. Immunohistochemical staining of P450 in the camel liver, kidney and brain confirmed the higher expression of P450 enzyme proteins in the liver and kidney as compared to other extrahepatic tissues. The maximum expression of P450 in the liver was observed in hepatocytes around the central vein and in the kidney it was observed in the proximal tubules. These results demonstrate that the multiple forms of P450s are differentially expressed in camel tissues and that the relative levels of expression are comparable with those of rat and human tissues. These observations may be important in understanding the differential susceptibility of camel tissues to the toxic/therapeutic effects of xenobiotics/drugs and environmental pollution.

Animals↗

PubMed1998

Differential response of cytosolic, microsomal, and mitochondrial glutathione S-transferases to xenobiotic inducers.

Subcellular levels of different isoenzymes of glutathione S-transferases (GSTs) and their catalytic activities in rat liver, lung and brain tissues were compared following treatment with phenobarbital (PB), -naphthoflavone (BNF) and dexamethasone (DEX). The constitutive expression of á and mu classes of GSTs, but not the GST , was maximum in the liver cytosol as compared to other tissues. Cytosolic GST activity using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate was 2-4 fold higher than that in the microsomal and mitochondrial fractions. Glutathione peroxidase activity with cumene hydroperoxide as a substrate was also highest in the rat liver cytosol. PB and BNF treatments markedly induced the amount of GST proteins in all the tissues studied with the maximum induction in the cytosol after 4 days of PB and 10 days of BNF treatments, respectively. The longer duration of treatments had a suppressive effect on the GST activity, particularly in the mitochondrial and microsomal fractions. DEX treatment, on the other hand, only marginally induced the cytosolic GST, while the mitochondrial GST and the membrane bound microsomal GST activities were mostly decreased. Northern blot analysis also showed an increase in the GST-á mRNA level indicating a possible upregulation of the GST gene expression by the xenobiotic agent. Differences between the subcellular GSTs were studied by the in vitro addition of N-ethylmaleimide (NEM), a selective activator of the microsomal GST. The cytosolic GST activity, both in livers of uninduced and PB-treated, was inhibited to about 50% of the control levels by NEM. The mitochondrial activity, on the other hand, was significantly activated by the addition of NEM, similar to that reported for the microsomal GST. These results suggest selectivity in the effects of different xenobiotics on the expression and catalytic activity of GST isoenzymes from different subcellular compartments of tissues. More importantly, these observations are also relevant in studies on xenobiotic induced organ-specific toxicity and carcinogenicity.

Animals↗

PubMed1997

Structural and functional aspects of rat microsomal glutathione transferase. The roles of cysteine 49, arginine 107, lysine 67, histidine, and tyrosine residues.

Rat liver microsomal glutathione transferase is rapidly inactivated upon treatment with the arginine-selective reagent phenylglyoxal or the lysine-selective 1,3,5-trinitrobenzenesulfonate. Glutathione sulfonate, an inhibitor of the enzyme, gives nearly complete protection against inactivation and prevents modification, indicating that these residues form part of or reside close to the active site. Sequence analysis of peptides from peptic and tryptic digests of [7-14C]phenylglyoxal- and 1,3,5-trinitrobenzenesulfonate-treated microsomal glutathione transferase indicated arginine 107 and lysine 67 as the sites of modification. A set of mutant forms of microsomal glutathione transferase was constructed by site-directed mutagenesis and heterologously expressed in Escherichia coli BL21(DE3). Arginine 107 was exchanged for alanine and lysine residues. The alanine mutant (R107A) exhibited an activity and inhibition profile similar to that of the wild type enzyme but displayed a decreased thermostability. Thus, arginine 107 does not appear to participate in catalysis or substrate binding; instead, an important structural role is suggested for this residue. Lysine 67 was mutated to alanine and arginine with no effect on activity. All three histidines were replaced by glutamine, and the resulting mutant proteins had activities comparable with that of the wild type. It can thus be concluded that the chemical modification experiments indicating that arginine 107, lysine 67, and one of the histidines partake in catalysis can be disproved. However, protection from modification by a competitive inhibitor indicates that these residues could be close to the glutathione binding site. All tyrosine to phenylalanine substitutions resulted in mutants with activities similar to that of the wild type. Interestingly, the exchange of tyrosine 137 appears to result in activation of the enzyme. Thus, the microsomal glutathione transferase must display an alternate stabilization of the thiolate anion of glutathione other than through interaction with the phenolic hydroxyl group of a tyrosine residue. Substitution of cysteine 49 with alanine resulted in a semiactivated mutant enzyme with enzymatic properties partly resembling the activated form of microsomal glutathione transferase. The function of this mutant was not altered upon reaction with N-ethylmaleimide, and cysteine 49 is thus demonstrated as the site of modification that results in activation of microsomal glutathione transferase.

Amino Acid Sequence↗

PubMed1996

Effect of bitter melon (Momordica charantia) fruit juice on the hepatic cytochrome P450-dependent monooxygenases and glutathione S-transferases in streptozotocin-induced diabetic rats.

Bitter melon (Momordica charantia), commonly known as karela, has been reported to have hypoglycemic, antiviral, antidiabetic, and antitumor activities. In the present study, we have investigated the effects of oral feeding of karela fruit juice on the hepatic cytochrome P450 (CYP) and glutathione S-transferase (GST) drug-metabolizing enzymes in the streptozotocin (STZ)-induced diabetic rat. Hepatic CYP contents, ethoxycoumarin-O-deethylase (ECOD), ethoxyresorufin-O-deethylase (EROD), aniline hydroxylase (AH), and aminopyrene N-demethylase (APD) activities were measured in control, diabetic, and karela juice fed animals. Diabetic rats exhibited a 50-100% increase in AH and EROD activities that was reversed by karela juice feeding. In addition, a decrease (17-20%) in the activities of APD and ECOD was observed in diabetic rat liver. Feeding of karela juice to the diabetic animals brought the level of APD close to that of control animals, while ECOD was further reduced to 60% of the control value. The cytosolic glutathione concentration was decreased in diabetic rats, and karela juice feeding normalized the effect. However, an increase (of 20-30%) in the GST activity was observed in both diabetic and karela juice fed rats. Western immunoblot analysis of CYP and GST isozymes exhibited a differential response during diabetes. The expression of CYP1A1, 2B1, 2E1, 3A4, and 4A2 in diabetes, while a decrease in GST mu was observed. Our results suggest that the changes in hepatic phase I and phase II drug-metabolizing enzyme activities in the STZ-induced diabetic animals may be associated with the altered expression of different CYP and GST isozymes. In addition, we have also observed that karela does not always reverse the effects on drug-metabolizing enzymes in STZ-induced diabetes.

Animals↗

PubMed1996

Membrane topology of recombinant rat liver microsomal glutathione transferase expressed in E. coli.

Rat liver microsomal glutathione transferase is a mammalian membrane protein that can be successfully expressed in Escherichia coli in an enzymatically active form. The protein does not form inclusion bodies and is recovered in the membrane fraction. The membrane topology of recombinant rat liver microsomal glutathione transferase expressed in E. coli was investigated by comparing the proteolytic cleavage products from intact and permeabilized spheroplasts. It was shown that lysine-4 of microsomal glutathione transferase is directed towards the outside, whereas lysine-41 faces the inside of the E. coli inner membrane. This shows that microsomal glutathione transferase has an inside-out orientation in E. coli spheroplasts as compared to liver microsomes. This fact enables us to make topology experiments that were previously not possible. Intact spheroplasts treated with pronase yielded a cleavage pattern consistent with two additional exposed segments closer to the C-terminus. Thus a polytopic model is suggested for the membrane association of microsomal glutathione transferase.

Animals↗

PubMed1996

Metabolism of 2-amino-alpha-carboline. A food-borne heterocyclic amine mutagen and carcinogen by human and rodent liver microsomes and by human cytochrome P4501A2.

2-Amino-alpha-carboline (A alpha C) is a mutagenic and carcinogenic heterocyclic amine that is formed as a pyrolysis product during the high temperature cooking of food and the burning of tobacco. Human, rat, and mouse hepatic microsomes each catalyzed the NADPH-dependent oxidation of A alpha C to form six products separable by HPLC. The two major metabolites, which together accounted for approximately 85% of the total metabolism, were characterized by UV, fluorescence, proton magnetic resonance, and mass spectral analyses as 3-hydroxy-A alpha C and 6-hydroxy-A alpha C. The remaining 15% were judged to be N-hydroxy-A alpha C and its oxidation products, based on chromatographic and spectral comparisons with a standard, whose synthesis and characterization are also described. Although the proportions of each metabolite were similar across species and individuals, the overall rate of metabolism of A alpha C by human hepatic microsomes showed a wide interindividual variation (37-fold), with a mean activity that was comparable with that observed with rat or mouse liver microsomes. alpha-Naphthoflavone, a selective inhibitor for cytochromes P4501A1 and P4501A2, strongly inhibited formation of both ring-hydroxylation and N-oxidation products by human, rat, or mouse liver hepatic microsomes. In addition, A alpha C oxidation was strongly correlated (r = 0.98; p < 0.001) with the oxidation of 4-aminobiphenyl, a known selective substrate for human and rodent cytochromes P4501A2. Immunoblot analyses confirmed the presence of cytochromes P4501A2, and not P4501A1, in human liver microsomes. Additional studies using recombinant human cytochromes P450 show that high catalytic activity for A alpha C metabolism was associated with human cytochrome P4501A2. Lower, but significant activity was also noted for P4501A1 and P4502C10, which could have important implications for the metabolic activation of A alpha C extrahepatic tissues. Neither A alpha C metabolism nor immunoreactive cytochrome P4501A2 (or P4501A1) was detected in human pancreatic microsomes. Although further carcinogenicity and biomarker studies for A alpha C are needed, the high rate of A alpha C metabolism by human liver cytochrome P4501A2 suggests that humans with the rapid P4501A2 phenotype with may be more susceptible than rodents to this heterocyclic amine carcinogen.

Animals↗

PubMed1995

beta-Naphthoflavone-inducible cytochrome P4501A1 activity in liver microsomes of the marine safi fish (Siganus canaliculatus).

The cytochrome P450-dependent metabolism of benzo(a)pyrene and other xenobiotics has been investigated in liver microsomes prepared from a local marine safi fish, Siganus canaliculatus. The safi fish was found to have a well-developed microsomal monooxygenase system consisting of cytochrome P450, cytochrome b5 and NADPH-cytochrome c reductase. The fish microsomal enzyme system was able to metabolize benzo(a)pyrene, 7-ethoxycoumarin and 7-ethoxyresorufin. Male fish were found to exhibit a higher monooxygenase activity than female fish. Treatment of fish with beta-naphthoflavone was found to induce (2- to 4-fold) the activities of aryl hydrocarbon hydroxylase, ethoxycoumarin O-deethylase and ethoxyresorufin O-deethylase. HPLC analysis of the metabolites produced by incubation of benzo(a)pyrene with the liver microsomal preparation showed a predominant formation of 3-OH and 9-OH benzo(a)pyrene. There was an increased formation of benzo(a)pyrene 7,8-diol and benzo(a)pyrene 7,8,9,10-tetrol in liver microsomes prepared from beta-naphthoflavone-treated fish. Western immunoblot analysis of liver microsomes from beta-naphthoflavone-treated fish using an antibody to rat liver cytochrome P4501A1 (CYP1A1) suggested the presence of an inducible cytochrome P450 enzyme that was comparable with that of rat liver enzyme. Our results suggest that liver microsomes from the safi fish have multiple forms of cytochrome P450 with a specific beta-naphthoflavone-inducible CYP1A1 homologous protein that can metabolize a variety of substrates.

Animals↗

PubMed1995

Alteration of glutathione, glutathione S-transferase and lipid peroxidation in mouse skin and extracutaneous tissues after topical application of gasoline.

The skin is the major environmental interface of the human body and is repeatedly exposed to a broad array of exogenous chemicals potentially capable of causing toxicity. In the present study we have applied 3, 6 or 12 ml leaded gasoline/kg body weight to the skin of adult male Swiss mice for 7 consecutive days and then sacrificed the animals on 8th day after an overnight fast. Glutathione (GSH) concentration, lipid peroxidation and other GSH-dependent enzyme activities were measured in skin, liver, brain and blood tissues of the mice. Topical application of 12 ml/kg gasoline caused a significant increase in water consumption by the animals, although, their body weight and food consumption was not significantly affected. A 40-60% decrease in blood concentration of glucose, triglyceride, and cholesterol was also observed after the treatment. The hemoglobin concentration, GSH content, lipid peroxidation and glucose 6-phosphate dehydrogenase activity of erythrocytes were not significantly affected by the gasoline treatment. However, a decrease in GSH concentration (16-21%), lipid peroxidation (30-60%) and glutathione S-transferase (GST) activity (30-40%) was observed in skin, liver and brain after gasoline application. Western blot analysis of tissues using antibodies against GST isoenzymes demonstrated an alteration in the expression of various GST isoenzymes after gasoline treatment. Our results suggest that topical exposure of gasoline causes some deleterious effects on skin and extracutaneous tissues.

Animals↗