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Considering the biological significance of 1,3,4-thiadiazole/oxadiazole heterocyclic scaffolds, a novel series of 1,3,4-thiadiazole-1,3,4-oxadiazole-acetamide derivatives (7a-j) was designed and synthesized using molecular hybridization. The inhibitory effects of the target compounds on elastase were evaluated, and all of these molecules were found to be potent inhibitors compared to the standard reference oleanolic acid. Compound 7f exhibited the excellent inhibitory activity (IC 50  = 0.06 ± 0.02 μM), which is 214-fold more active than oleanolic acid (IC 50  = 12.84 ± 0.45 μM). Kinetic analysis was also performed on the most potent compound (7f) to determine the mode of binding with the target enzyme, and it was discovered that 7f inhibits the enzyme in a competitive manner. Furthermore, the MTT assay method was used to assess their toxicity on the viability of B16F10 melanoma cell lines, and all compounds did not display any toxic effect on the cells even at high concentrations. The molecular docking studies of all compounds also justified with their good docking score and among them, compound 7f had a good conformational state with hydrogen bond interactions within the receptor binding pocket, which is consistent with the experimental inhibition studies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Herein, we have synthesized quinoline united various Schiff base derivatives (Q1-Q13) and systematically characterized them using diverse analytical practices such as 1 H NMR, 13 C NMR, FT-IR and LC-MS respectively. All of the target compounds that have been synthesized were tested for elastase inhibition, and the findings were compared to the standard drug oleanolic acid. Among the entire series, compound Q11 (IC 50  = 0.897 ± 0.015 µM) exhibit most promising elastase inhibitory activity than oleanolic acid (Standard) having an IC 50 value of 13.426 ± 0.015 µM. Also, the utmost effectivecompound Q11 was used for kinetic mechanism investigation based on in-vitro data, from which it has been concluded that compound Q11 inhibits elastase competitively. Furthermore, utilizing the MTT test approach, the most effective compounds were assessed for cytotoxicity on B16F10 melanoma cells. From the cytotoxicity experiment, the most potent compound did not display any hazardous response against B16F10 melanoma cells despite being treated at high concentrations. Additionally, the molecular docking study was settled to govern the binding interaction pattern among an enzyme and inhibitors., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Multi-scale structural interactions of the arrowhead starch-linoleic/stearic acid complexes under different durations (20, 40 & 60 min) of dual-frequency power ultrasound (DFPU, 20/40 kHz) and their underlying mechanisms were discussed. Differential scanning calorimetry and X-ray diffraction (XRD) revealed V6 type (V6-I, II) crystalline structure for ultrasonically-treated arrowhead starch-linoleic acid (UTAS-LA) complexes. An increased degree of short-range molecular order as IR ratios of 1045/1022 cm -1 was evident from the FTIR results. The complexing index (CI) values of the complexes were greater than 65%, and the highest CI values of 83.04% and 81.26% were found in the case of UTAS-LA40 and UTAS-LA60, respectively. SEM results showed that LA-complexes had a sponge-like structure with smooth surfaces, while the SA-complexes exhibited flaky structures with irregular shapes and rough surfaces. The V-type complexes exhibited a higher digestion resistance than native AS and un-sonicated AS-LA/SA complexes due to partial RDS convention to RS., (Copyright © 2022. Published by Elsevier B.V.)

Sagittaria sagittifolia L. is a well-known plant, belongs to the Alismataceae family. Sonication can improve the functional properties of starch; hence, the aim of this study was to develop ultrasonically modified arrowhead starch (UMAS) using a sophisticated and eco-friendly tri-frequency power ultrasound (20/40/60 kHz) method at 300, 600, and 900 W for 15 and 30 min. Significant (p < 0.05) increases in swelling power, solubility, and water and oil holding capacities were achieved. FTIR spectroscopy corroborated the ordered, amorphous, and hydrated crystals of the sonicated samples. Increases in sonication frequency and power led to significant (p < 0.05) increases in onset gelatinization temperatures. Scanning electron microscopic analysis of sonicated samples showed superficial cracks and roughness on starch granules appeared in a sonication power-dependent manner compared with that of untreated sample. Overall, the ultrasonically-treated samples showed improved physicochemical properties, which could be useful for industrial applications., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Ultrasound treatment was used to successfully prepare Quercetin (Qu)-loaded Casein phosphopeptides (CPP)/chitosan (CS) nanoparticles. Compared with the control, the above ternary nanoparticles with the smallest size (241.27 nm, decreased by 34.32%), improved encapsulation efficiency of Qu (78.55%, increased by 22.12%) when prepared under following conditions: ultrasonic frequency, 20/35/50 kHz; the power density, 80 W/L; the time, 20 min, and the intermittent ratio, 20 s/5s. Electrostatic interactions, hydrogen bonding, and hydrophobic interactions were the main driving forces for nanoparticles formulation, which were strengthened by ultrasound treatment. The compact, homogeneous and spherical composite nanoparticles obtained by sonication were clearly observed by scanning electron microscope and atomic force microscope. The environmental stability (NaCl, pH, exposure time, storage time, and simulated gastrointestinal digestion) and antioxidant activity of the ternary nanoparticles were remarkably enhanced after ultrasonic treatment. Furthermore, the ternary nanoparticles prepared by ultrasound exhibited excellent stability in simulated gastrointestinal digestion. The above results indicate that ultrasound not only increases the loading of the nanoparticles on bioactive substances but also improves the environmental stability and antioxidant activity of the formed nanoparticles. Ultrasound-assisted preparation of nanoparticles loaded with bioactive substances could be well used in the functional food and beverage industry., (Copyright © 2021. Published by Elsevier B.V.)

In this research work, we have designed and synthesized some biologically useful of 1,3,4-Oxadiazoles. The structural interpretation of the synthesized compounds has been validated by using FT-IR, LC-MS, HRMS, 1 H NMR and 13 C NMR techniques. Moreover, the in-vitro mushroom tyrosinase inhibitory potential of the target compounds was assessed. The in-vitro study reveals that, all compounds demonstrate an excellent tyrosinase inhibitory activity. Especially, 2-(5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-ylthio)-N-phenylacetamide (IC 50  = 0.003 ± 0.00 µM) confirms much more significant potent inhibition activity compared with standard drug kojic acid (IC 50  = 16.83 ± 1.16 µM). Subsequently, the most potent five oxadiazole compounds were screened for cytotoxicity study against B16F10 melanoma cells using an MTT assay method. The survival rate for the most potent compound was more pleasant than other compounds. Furthermore, the western blot results proved that the most potent compound considerably decreased the expression level of tyrosinase at 50 µM (P < 0.05). The molecular docking investigation exposed that the utmost potent compound displayed the significant interactions pattern within the active region of the tyrosinase enzyme and which might be responsible for the decent inhibitory activity towards the enzyme. A molecular dynamic simulation experiment was presented to recognize the residual backbone stability of protein structure., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or COVID-2019 is a new global health challenge which causes severe respiratory complications. As of May 17th, 2020, SARS-CoV-2 has infected 4.6 million people and caused 310,000 deaths, worldwide. In order to study potential impact of infection, complete epidemiological information should be reported on regular basis however, data from Pakistan has not yet been published. This retrospective study is the first report of epidemiological trends of COVID-19 in Faisalabad, Pakistan. On April 4th, 2020, 128 nasopharyngeal swabs collected from city Faisalabad were transported to Postgraduate Research Institute, Lahore for further processing. RNA was extracted using QIAsymphony DSP Virus/Pathogen Midi Kit and real-time PCR was performed to quantify COVID-19. Our finding showed that overall prevalence of COVID-19 in Faisalabad on April 4th was 17.18% (22 of 128). Prevalence was higher in males (n = 17; 77.2%) as compared to females (n = 5; 22.8%) but this gender-wise difference was not statistically significant. Patients belonging to age group 37-47 years were found to be most (45.5%) infected with COVID-19., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020. Published by Elsevier B.V.)

The present article describes the synthesis, in vitro urease inhibition and in silico molecular docking studies of a novel series of bi-heterocyclic bi-amides. The synthesis of title compounds was initiated by benzoylation, with benzoyl chloride (1), of the key starter ethyl 2-(2-amino-1,3-thiazol-4-yl)acetate (2) in weak basic aqueous medium followed by hydrazide formation, 4, and cyclization with CS 2 to reach the parent bi-heterocyclic nucleophile, N-{4-[(5-sulfanyl-1,3,4-oxadiazol-2-yl)methyl]-1,3-thiazol-2-yl}benzamide (5). Various electrophiles, 8a-l, were synthesized by a two-step process and these were finally coupled with 5 to yield the targeted bi-heterocyclic bi-amide molecules, 9a-l. The structures of the newly synthesized products were corroborated by IR, 1 H NMR, 13 C NMR, EI-MS and elemental analysis. The in vitro screening of these molecules against urease explored that most of the compounds exhibit potent inhibitory potential against this enzyme. The compound 9j, with IC 50 value of 2.58 ± 0.02 µM, exhibited most promising inhibitory activity among the series, relative to standard thiourea having IC 50 value of 21.11 ± 0.12 µM. In silico studies fully augmented the experimental enzyme inhibition results. Chemo-informatics analysis showed that synthesized compounds (9a-l) mostly obeyed the Lipinski's rule. Molecular docking study suggested that ligand 9j exhibited good binding energy value (-7.10 kcal/mol) and binds within the active region of target protein. So, on the basis of present investigation, it was inferred that 9j may serve as a novel scaffold for designing more potent urease inhibitors., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

To seek the new medicinal potential of sulfadiazine drug, the free amino group of sulfadiazine was exploited to obtain acyl/aryl thioureas using simple and straightforward protocol. Acyl/aryl thioureas are well recognized bioactive pharmacophore containing moieties. A new series (4a-4j) of sulfadiazine derived acyl/aryl thioureas was synthesized and characterized through spectroscopic and elemental analysis. The synthesized derivatives 4a-4j were subjected to calf intestinal alkaline phosphatase (CIAP) activity. The derivative 4a-4j showed better inhibition potential compared to standard monopotassium phosphate (MKP). The compound 4c exhibited higher potential in the series with IC 50 0.251 ± 0.012 µM (standard KH 2 PO 4 4.317 ± 0.201 µM). Lineweaver-Burk plots revealed that most potent derivative 4c inhibition CIAP via mixed type pathway. Pharmacological investigations showed that synthesized compounds 4a-4j obey Lipinsk's rule. ADMET parameters evaluation predicted that these molecule show significant lead like properties with minimum possible toxicity and can serve as templates in drug designing. The synthetic compounds show none mutagenic and irritant behavior. Molecular docking analysis showed that compound 4c interacts with Asp273, His317 and Arg166 amino acid residues., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

It is widely accepted that non-steroidal anti-inflammatory drugs (NSAIDs), including aspirin, reduce the risk of cancer. The anti-cancer and anti-inflammatory effects of NSAIDs are associated with the inhibition of prostaglandin synthesis and cyclooxygenase-2 activity. Several other mechanisms which contribute to the anti-cancer effect of these drugs in different cancer models both in vivo and in vitro are also presumed to be involved. The precise molecular mechanism, however, is still not clear. We investigated, therefore, the effects of acetylsalicylic acid (ASA, aspirin) on multiple cellular and functional targets, including mitochondrial bioenergetics, using human hepatoma HepG2 cancer cells in culture. Our results demonstrate that ASA induced G0/G1 cell cycle arrest and apoptosis in HepG2 cells. ASA increased the production of reactive oxygen species, reduced the cellular glutathione (GSH) pool and inhibited the activities of the mitochondrial respiratory enzyme complexes, NADH-ubiquinone oxidoreductase (complex I), cytochrome c oxidase (complex IV) and the mitochondrial matrix enzyme, aconitase. Apoptosis was triggered by alteration in mitochondrial permeability transition, inhibition of ATP synthesis, decreased expression of the anti-apoptotic protein Bcl-2, release of cytochrome c and activation of pro-apoptotic caspase-3 and the DNA repairing enzyme, poly (-ADP-ribose) polymerase (PARP). These findings strongly suggest that ASA-induced toxicity in human hepatoma HepG2 cells is mediated by increased metabolic and oxidative stress, accompanied by mitochondrial dysfunction which result in apoptosis., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Peptide-based defenses of ranid frogs from Mexico and Central America have been studied in much less detail than those from North America. Peptides belonging to the brevinin-1 (5 peptides), palustrin-2 (1 peptide), and ranatuerin-2 (3 peptides) families were isolated from norepinephrine-stimulated skin secretions of the Costa Rican frog, Lithobates vaillanti (Ranidae) and characterized structurally. Brevinin-1VLa (FLGAIAGVAAKFLPKVFCFITKKC) and brevinin-1VLc (FLPVIASVAAKVLPK VFCFITKKC) showed particularly high growth-inhibitory potency (MIC < or =3 microM) against a Gram-positive microorganism Staphylococcus aureus and the opportunistic yeast pathogen Candida albicans and potent cytolytic activity (LC(50)< or =8 microM) against both human erythrocytes and HepG2 hepatoma-derived cells. The peptides were also active against a Gram-negative microorganism Escherichia coli (MIC< or =50 microM). Substitutions in brevinin-1VLd (Lys(11) --> Asn) and brevinin-1VLe (Lys(11) --> Ser) that decrease cationicity result in loss of activity against E. coli. Ranatuerin-2VLb (GIMDTIKGAAKDLAGQLLDKLKCKITKC) showed relatively weak antimicrobial activity (MIC> or =75 microM) but selective cytolytic activity against HepG2 tumor cells (LC(50)=30 microM) compared with erythrocytes (LC(50)>200 microM). In addition, a dodecapeptide (RICYAMWIPYPC) were isolated from the secretions that were devoid of antimicrobial activity. This component contains an Ala-Met bond that constitutes the scissile bond in the selective elastase inhibitor, elafin but the peptide did not inhibit pancreatic elastase at concentrations up to 100 microM.

Background: There is an independent association between white blood cell (WBC) and coronary heart disease (CHD) risk. However, the relationship between WBC and Framingham Risk Score (FRS) remains unclear., Methods: This is a cross-sectional study on a consecutive sample of 520 white asymptomatic men (mean age 46 +/- 7 years) without CHD. The study population was divided into WBC quartiles (x10(9) cells/L): 1(st) quartile: 3.1-5.3 (n = 139), 2(nd) quartile: 5.4-6.1 (n = 129), 3(rd) quartile: 6.2-7.1 (n = 131), 4(th) quartile: >/=7.2 (n = 121), and into tertiles according to the 10-year FRS: 1(st) tertile (low risk <5%, n = 180, 35%), 2(nd) tertile (intermediate risk 5-12%, n = 210, 40%), 3(rd) tertile (high risk: >/=13%, n = 130, 25%)., Results: WBC correlated only weakly with FRS (r = 0.18, p = 0.001). Among individual components of FRS, WBC correlated minimally with smoking (r = 0.12, p = 0.003), systolic blood pressure (r = 0.07, p = 0.1), and high-density lipoprotein cholesterol (r = -0.06, p = 0.1). However, no correlation was observed with age (p = 0.3) and total cholesterol (p = 0.5). Nearly one third (31%) of men in the low-risk (FRS <5%) had WBC count in the 1(st) quartile compared to 20% of those classified as high risk (FRS >/=13%). The prevalence of WBC in the 4(th) quartile increased across FRS tertiles (18, 22, 32%) (p = 0.09)., Conclusions: WBC correlates weakly with FRS or its individual components. Since WBC count is strongly related to CHD, WBC may reflect different components of cardiovascular risk, which might not be captured by traditional cardiovascular risk factors used in calculating FRS. Inflammatory biomarkers afford adjunctive value to FRS and may be used to improve CHD risk stratification.

Background: From a preventive aspect, it is especially important to investigate the lifestyle risk factors associated with cardiovascular disease (CVD). The purpose of this study was to determine the relationship of increasing metabolic syndrome (MS) components across increasing levels of estimated cardiorespiratory fitness (CRF) in asymptomatic young to middle-aged men., Methods: We studied 449 consecutive asymptomatic men (47 +/- 7 years) who underwent a maximal treadmill exercise test according to the Bruce protocol. Cardiorespiratory fitness (CRF) was divided into tertiles based on metabolic equivalents (METs). The following MS components were studied: 1) waist circumference > 102 cm; 2) serum triglycerides > or = 150 mg/dL; 3) HDL cholesterol levels of < 40 mg/dL; 4) fasting blood glucose (FBG) > or = 110 mg/dL or 5) blood pressure > or = 130/85 mmHg or treated hypertension. Multinomial logistic regression was used to investigate the relationship between clustering of MS components and CRF as determined by metabolic equivalents (METs). We used polytomous logistic regression to determine the likelihood of clustering of increasing components of metabolic syndrome with intermediate (2nd tertile) and low (1st tertile) levels of CRF as compared to those with highest levels of CRF (3rd tertile)., Results: Overall in the study population, zero, 1, 2 and > or = 3 (i.e., metabolic syndrome) risk factors for MS were observed in 29% (n = 129), 26% (n = 118), 22% (n = 98) and 23% (n = 104) men, respectively. The mean METS achieved in the study population was 10 +/- 2 (range 4-20). Nearly half (49%) of individuals with the highest levels of CRF had no MS risk factors whereas only 18% of those with low CRF (METS < 9) had no MS risk factors. On the other end of the spectrum, the prevalence of MS (> or = 3 MS risk factors) increased significantly across decreasing levels of CRF (6, 22, 33% p < 0.0001 for trend). Multivariable polytomous logistic regression (adjusting for age, smoking, cholesterol-lowering therapy) demonstrated that individuals with low CRF (1st tertile of METS) compared to those with highest CRF had 3.1- (p = 0.001) and 11.8- (p < 0.0001) fold higher risk of having 2 and > or = 3 MS components, respectively. Similar results were observed when the analyses was repeated adjusting for Framingham risk score., Conclusions: Asymptomatic men with low levels of CRF have a greater likelihood for clustering of MS components and thus are at higher CVD risk. Further studies are needed to define the risk of cardiovascular disease in patients with intermediate levels of CRF and address which treatment strategies are most important given an individual's risk profile.

We previously reported the occurrence of multiple forms of drug metabolizing enzymes in camel tissues. In this study, we demonstrated for the first time, flavin-containing monooxygenase (FMO)-dependent metabolism of two model substrates methimazole (MEM) and N,N'-dimethylaniline (DMA) by camel liver, kidney, brain and intestine. FMO-catalyzed metabolism in the microsomes of camel tissues was independent of cytochrome P450 (CYP) activity and exhibited a pH and temperature dependence characteristic of FMO enzymes. Use of inhibitors of CYP activities, SKF525A, octylamine or antibody against NADPH-P450 reductase, did not significantly alter the FMO-dependent substrate metabolism. Using MEM as a model substrate for FMO activity, we show that camel liver has an activity similar to that in rat and human livers. MEM metabolism in extrahepatic tissues in camels was significantly lower (60%-80%) than that in liver. Our results suggest occurrence of FMO in camel tissues, with catalytic properties similar to those in rat and human livers. These results may help in better understanding the effects of pharmacologically and toxicologically active compounds administered to camels.

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.

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.

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.

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.

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.

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.

1. Previous studies have demonstrated the presence of phase I mixed-function oxidases (cytochrome P450-dependent) and phase II conjugation (glutathione S-transferase) enzymes in camel liver. This study represents further characterisation of these drug metabolising enzyme systems in camel liver by comparing their catalytic and immunochemical properties with enzymes of rat and mouse liver. 2. Using the specific P450 substrate aniline, the microsomal aniline hydroxylase activity of camel liver was found to be significantly lower than that of rat and mouse. The Km values of the enzyme for aniline was similar in rat and camel liver; however, the Vmax for camel liver enzyme was 50% of the rat liver enzyme. Aminopyrene N-demethylase activity in camel liver, was lower than that of rat but higher than in mouse. Microsomal NADPH cytochrome C-reductase and NADPH-supported lipid peroxidation activities were similar in all three species. 3. The cytosolic phase II conjugation enzyme glutathione S-transferase and glutathione peroxidase activities in camel liver were markedly lower than those of rat and mouse enzymes. However, GSH concentration was similar in all three species. 4. Immunodot blot and Western blot analysis of liver cytosols, using antibodies to specific GST isoenzymes, have shown that camel liver like mouse and rat, expresses predominantly the Alpha and Mu classes of GST. GST Pi on the other hand, was abundant in mouse liver and was underexpressed in camel and rat liver. 5. Our results demonstrate that there are multiple forms of phase I (P450) and phase II (GST) enzymes in camel liver and that they are comparable with the drug metabolising enzymes of rat and mouse.(ABSTRACT TRUNCATED AT 250 WORDS)

The glutathione S-transferase (GST)-dependent conjugation of reduced glutathione (GSH) with leukotriene A4 (LTA4)-methyl ester in rodent and human skin was investigated. Incubation of [3H]LTA4-methyl ester (1 nmole, approximately 200,000 dpm) with cytosol prepared from rat, mouse and human skin or with affinity purified GST from rat skin cytosol in the presence of GSH resulted in the formation of LTC4-methyl ester. Maximum enzyme activity was observed in rat skin followed by mouse and human skin. With heat-denatured cytosol or in the absence of GSH, the product formation was negligible. GST purified from rat skin cytosol by GSH-agarose affinity chromatography exhibited a several-fold increase in the specific activity of enzyme with 1-chloro-2,4-dinitrobenzene (55-fold), ethacrynic acid (67-fold) and LTA4-methyl ester (12-fold) as substrates. Western blot analysis of the affinity purified GST indicated a predominant expression of the Pi class of GST isozyme followed by Mu and Alpha classes of isozymes. The formation of LTC4-methyl ester was established by its radioactivity profile on high pressure liquid chromatography and absorption spectroscopy. These results suggest that, in addition to xenobiotic metabolism, cutaneous GSTs may also be capable of metabolizing physiological substrates such as LTA4.