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The present study investigated the effects of dietary oltipraz on cigarette smoke-related lipophilic DNA adduct formation. Female Sprague-Dawley rats were exposed daily to sidestream cigarette smoke in a whole-body exposure chamber 6 h/day for 4 consecutive weeks. One group of rats was maintained on control diet while another group received the same diet supplemented with either a low (167 p.p.m.) or high (500 p.p.m.) dose of oltipraz, starting 1 week prior to initiation of smoke exposure until the end of the experiment. Analysis of lipophilic DNA adducts by the nuclease P1-mediated 32P-post-labeling showed up to five smoke-related adducts. Adduct no. 5 predominated in both the lung and the heart while adduct nos 3 and 2 predominated in the trachea and bladder, respectively. Quantitative analysis revealed that the total adduct level was the highest in lungs (270 ± 68 adducts/1010 nucleotides), followed by trachea (196 ± 48 adducts/1010 nucleotides), heart (141 ± 22 adducts/1010 nucleotides) and bladder (85 ± 16 adducts/1010 nucleotides). High dose oltipraz treatment reduced the adduct levels in lungs and bladder by >60%, while the reduction in lungs in the low-dose group was 35%. In trachea, the effect of low and high dietary oltipraz on smoke DNA adduction was equivocal, while smoke-related DNA adducts in the heart were minimally inhibited by high-dose oltipraz. In a repeat experiment that employed a 3-fold lower dose of cigarette smoke, oltipraz (500 p.p.m.) was found to inhibit the formation of DNA adducts in rat lungs and trachea by 80 and 65%, respectively. These data clearly demonstrate a high efficacy of oltipraz in inhibiting the formation of cigarette smoke-induced DNA adducts in the target tissues. [ABSTRACT FROM PUBLISHER]

The research aims to identify the inhibitory potential of natural dietary phytochemicals against non-insulinotropic target protein alpha-glucosidase and its possible implications to diabetes mellitus type 2. A data set of sixteen plant-derived dietary molecules viz., 4,5-dimethyl-3-hydroxy-2(5H)-furanone, apigenin, bromelain, caffeic acid, cholecalciferol, dihydrokaempferol 7-o-glucopyranoside, galactomannan, genkwanin, isoimperatorin, luteolin, luteolin 7-o-glucoside, neohesperidin, oleanoic acid, pelargonidin-3-rutinoside, quercetin, and quinic acid were taken to accomplish molecular docking succeeded by their comparison with known inhibitors including acarbose, miglitol, voglibose, emiglitate, and 1-deoxynojirimycin. Among all phyto-compounds, bromelain (ΔG: -9.54 kcal/mol), cholecalciferol (-8.47 kcal/mol), luteolin (-9.02 kcal/mol), and neohesperidin (-8.53 kcal/mol) demonstrated better binding interactions with alpha-glucosidase in comparison to the best-known inhibitor, acarbose (ΔG: -7.93 kcal/mol). Molecular dynamics simulation of 10 ns duration, CYP450 site of metabolism identification, and prediction of activity spectra for substances depicted the bromelain as the most stable inhibitor compared to luteolin and acarbose. Findings of molecular interactions, molecular dynamics study, metabolism, and biological activity prediction proved bromelain as a potential alpha-glucosidase inhibitor. Thus, bromelain might be helpful as an insulin-independent therapeutic molecule towards controlling and managing diabetes mellitus type 2.

Malware is a blanket term for Trojan, viruses, spyware, worms, and other files that are purposely created to harm computers, mobile devices, or computer networks. Malware commonly steals, encrypts, damages, and causes a mess in these devices. The growth of malware attacks has a consequence on the growth and attractiveness of mobile features in mobile devices. Most malware research aims to probe the different methods of preventing, analysing, and detecting malware attacks. This paper aims to demonstrate an exhaustive knowledge map of the Android malware by collecting a ten (10) year dataset from the Web of Science database. A bibliometric analysis was employed for analysing articles published between 2010 and 2019. Using the keyword "malware", 5622 articles were retrieved. After scrutinising with the keywords of "Android malware", 1278 articles were then collected. This study provides an overview of the articles, productivity, research area, the Web of Science categories, authors, high-cited articles, institutions, and impact journals examining malware. Research activities are continued by placing terms in the classification of malware detection systems that outline important areas in malware research. From the analysis, it can be concluded that the highest number of publications focusing on malware studies came from the continent of Asia. Additionally, this study discusses the challenges of malware studies in the recent research studies as well as the future direction., (© Akadémiai Kiadó, Budapest, Hungary 2021.)

The present study was accomplished to examine and compare the effect of specific antioxidant-rich oils on hyperglycemia, dyslipidemia, renal function markers and oxidative renal damage in diabetic rats for four weeks. Papaya (P), olive (O), fenugreek (Fe), bitter gourd (B) and fish (Fi) oils were used for this purpose. Streptozotocin (STZ) was injected intraperitoneally in a single dose to induce diabetes. All oils were given orally at a dose of 3g/kg for four weeks in respective group after induction of diabetes. After treatment with oils, blood was collected, and their kidneys were stored. The level of fasting blood glucose (FBG), glycated hemoglobin (HbA1c), total cholesterol (TC), triglycerides (TG), low-density lipoprotein-cholesterol (LDL-C) and very low-density lipoprotein-cholesterol (VLDL-C) increased while amylase and high-density lipoprotein cholesterol (HDL-C) level decreased in the diabetic rats. These changes were augmented by fenugreek, bitter gourd and olive oils treatment. Diabetic rats showed elevated renal function markers in serum, including, serum creatinine (Scr), blood urea nitrogen (BUN) and alkaline phosphatase (ALP), which were restrained significantly by fenugreek and bitter gourd oil treatment. Moreover, fenugreek and bitter gourd oils treatment significantly modulated the level of thiobarbituric reactive substances (TBARS), malonaldehyde (MDA) and catalase (CAT) in the kidney of diabetic rats. The histopathological examination also showed the protective effect of these oils. The study suggests that vegetable oils are effective in reducing hyperglycemia, dyslipidemia and renal damage related to the side effects of diabetes. Thus they may have therapeutic value for preventing diabetes side effects and may be included in oil diet treatment synergically. Thus, our data suggest that oils as potent antidiabetic agent and beneficial in the control of diabetes-related abnormalities such as hyperglycemia, dyslipidemia and renal damage of STZ induced rat model of type 2 diabetes. Our study also supports the suggestion that synergistic possibilities exist concerning the use of these oils in the treatment of diabetes mellitus.

Breast cancer is the leading cause of death among women worldwide. It is a multi-factorial disease caused by genetic and environmental factors. Vitamin D has been hypothesized to lower the risk of breast cancer via the nuclear vitamin D receptor (VDR). Genetic variants of these vitamin D metabolizing genes may alter the bioavailability of vitamin D, and hence modulate the risk of breast cancer. Materials and Methods: The distribution of Fok1 VDR gene (rs2228570) polymorphism and its association with breast cancer was analysed in a case–control study based on 125 breast cancer patients and 125 healthy females from North Indian population, using PCR-RFLP. An In silico exploration of the probable mechanism of increased risk of breast cancer was performed to investigate the role of single nucleotide polymorphisms (SNPs) in cancer susceptibility. Results: The Fok1 ff genotype was significantly associated with an increased risk of breast cancer (p=0.001; χ2=13.09; OR=16.909; %95 CI=2.20 - 130.11). In silico analysis indicated that SNPs may lead to a loss in affinity of VDR to calcitriol, and may also cause the impairment of normal interaction of liganded VDR with its heterodimeric partner, the retinoid X receptor (RXR), at protein level, thereby affecting target gene transcription. Conclusion: Breast cancer risk and pathogenesis in females can be influenced by SNPs. SNPs in VDR may cause alterations in the major molecular actions of VDR, namely ligand binding, heterodimerization and transactivation. VDRE binding and co-activator recruitment by VDR appear to be functionally inseparable events that affect vitamin D-elicited gene transcription. This indicates that breast cancer risk and pathogenesis in females may be influenced by SNPs., (Creative Commons Attribution License)

In the proposed work, we have explicated the mechanism of dibenzo[a,l]pyrene (DBP) and benzo[a]pyrene (BP) modulated cell proliferation by assessing the plausible binding with CASPASES, BAX, Bcl-2, MDM2, p53, p21, p16, CylinD1-CDK4 complex, CylinE1-CDK2 complex, H-Ras, K-Ras, BRCA1, and BRCA2 through exploiting the inherent potential of AutoDock Tools 4.0. In silico findings revealed that potent carcinogenic metabolites of DBP (e.g., (-)-anti-DBPDE and (+)-syn-DBPDE) and BP (e.g., (+)-anti-BPDE) exhibited better binding interactions to Caspase-9 than Caspase-8 and Caspase-3. Feeble interactions of BAX and Bcl-2 with diol-epoxides of both PAHs were observed. Diol-epoxides of DBP and BP were found to bind to p53 with tighter interaction than MDM2 and p53-MDM2 complex. The p16 and Cyclin-CDK complexes were best docked to aforesaid metabolites as compared to p21. Moreover, stronger interactions of BRCA1 and BRCA2 with DBP and feeble interactions of BRCA1 and BRCA2 with BP were observed from docking results. Furthermore, stronger interactions of both DBP and BP with the H-Ras and K-Ras oncoproteins were found, while only DBP interacted relatively strongly with the BRCA1 and BRCA2, which were suggesting more carcinogenic nature of DBP than BP, a well-known observation in the wet lab. Besides giving structural insight into the mechanism of DBP and BP-mediated cell proliferation, these in silico findings may be helpful to understand the mechanistic nature of environmental carcinogens and their cellular targets.

Exposure to exogenous and endogenous chemicals and subsequent cellular and molecular changes has been linked to enhanced cell proliferation and restricted apoptosis phenomenon. Though in the past decades numerous anticancer drugs inducing programmed cell death in cancer cells by targeting specific apoptotic markers have reached the market, they have been allied with unwanted side effects, ranging from mild to severe toxicity. With further understanding on the functional mechanism of p53 and MDM2 in apoptosis and in our continuous search for new and potent multi-target anticancer lead compounds, we have carried out molecular docking and inhibition studies of the selected aglycones along with selected anticancer leads, against the specific apoptotic and cell cycle markers using AutoDock Tools 4.0 and other computational softwares. The docking results have been analyzed in terms of binding energies (kcal/mol) and inhibition constant (µM). The study clearly proposes our aglycones [solanidine (Solanid-5-en-3β-ol), solasodine (Solasod-5-en-3β-ol), and tomatidine (5α-Tomatidan-3β-ol)] induce apoptosis by inhibiting the p53-MDM2 complex, p21 Waf1/Cip1 , and Bcl-2 proteins, which were even found comparable with the anticancer drugs nutlin and/or halofuginone. The work further emphasizes that the individual molecular targets such as BAX and Bcl-2 may result in misleading data at any level; however, ratio of responses to BAX and Bcl-2 shall be considered for better clue about a compound to be pro- or anti-apoptotic.

To establish in silico model to predict the structural insight into the metabolic bioactivation pathway of xenobiotics, we considered two specific and one non-specific mammary procarcinogen [e.g., dibenzo[a,l]pyrene (DBP), 7,12-dimethylbenz[a]anthracene (DMBA), and benzo[a]pyrene (BP)]. The CYP1A1, 1B1, 2C9, 1A2 and 2B6 reported in wet-lab studies to actively metabolize DBP also showed strong binding energies (kcal/mol) of -11.50, -10.67, -10.37, -9.76 and -9.72, respectively, with inhibition constants ranging between 0.01 and 0.08 µM. The CYP3A4 depicted minimum binding energy (-9.51 kcal/mol) which is in agreement with the wet-lab reports. Further, relatively better affinity of CYP1A1 and CYP1B1 with the dibenzo[a,l]pyrene-11,12-diol (DBPD) might be indicative of their involvement in carcinogenicity of parent compound. Like DBP, BP (-10.13 kcal/mol, Ki: 0.04 µM) and BP-diols (BPD) (-9.01 kcal/mol, Ki: 0.25 µM) observed plausible binding with CYP1A1 supporting to the reported data that emphasize the major contribution of CYP1A1 in the activation of similar procarcinogens and mutagens. Likewise, in silico results further highlighted the CYP1A1 as key player in bioactivation of DMBA to its carcinogenic metabolites. In case of PhIP metabolism, strong binding interaction predicted with CYP1A1 (-9.63 kcal/mol) rather than CYP1A2 (-8.84 kcal/mol). Dissimilarity in the binding affinity of PhIP might be due to its basic scaffold. Further, molecular dynamics (MD) simulation of 10 ns has been revealed that docked complexes of CYP1A1 with DBP, DMBA and BP are comparatively more stable than the complex of PhIP. Moreover, the current findings might be valuable as reference model in prediction and elucidation of the approximate metabolic pathway of xenobiotics.

Background: Malignant tumors are the leading cause of death in humans. Due to the tedious efforts and investigations made in the field of marine drug discovery, there is now a scientific bridge between marine and pharmaceutical sciences. However, currently only few marine drugs have been lined towards anticancer direction yet many more to are be established in future as well., Method: This review gives an overview of present status of marine natural products MNPs both at the level of research and clinical stages. The authors haved summarized the detail information of diverse marine organisms that were reportedto be active or potentially active in cancer treatment in the last two decades. Interstingly, marine organisms are abundant producer of plenty of structurally incomparable bioactive metabolites that have unusual mode of actions and diverse biosynthetic pathways., Results: This review summarizes the associated anticancer properties of different classes of marine natural compounds based on their structural diversity, biological activity, and the molecular mechanisms of action. Emphasis has also be given to recent advances in clinical development of marine agents used in clinical trials., Conclusions: The present review is summarising the various sources of marine chemicals and their exploration of anticancerous potential. There is justified hope for the discovery and development of new anticancer agents from the marine environment., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Background: Designing a novel antagonist against VEGFR-2 is being applied currently to inhibit cancer growth and metastasis. Because of the unexpected side effects incurred by the contemporary anticancer medications, the focus has been laid towards identifying natural compounds that might carry the potential to inhibit tumor progression. VEGR-2 remains an important target for anticancer drug development as it is the master regulator of vascular growth., Objective: The study focuses on virtual screening of compounds from plants of Asteraceae family that bears antiangiogenic potential and thus, inhibiting VEGFR-2 using a computational approach., Materials and Methods: Structures of phytochemicals were prepared using ChemDraw Ultra 10 software and converted into its 3D PDB structure and minimized using Discovery Studio client 2.5. The target protein, VEGFR-2 was retrieved from RCSB PDB. Lipinski's rule and ADMET toxicity profiling were carried out on the phytochemicals of the Asteraceae family and the filtered compounds were further promoted for molecular docking and MD simulation analysis. The study extends towards the SOM analysis of Pinocembrin to predict the possible toxic and non-toxic in vivo metabolites via in silico tools (Xenosite Web and PASS online server)., Results: The docking results revealed promising inhibitory potential of Pinocembrin against VEGFR-2 with binding energy of -8.50 kcal/mole as compared to its known inhibitors Sorafenib and YLT192 having binding energy of -6.49 kcal/mole and -8.02 kcal/mol respectively. Further, molecular dynamics (MD) simulations for 10ns were conducted for optimization, flexibility prediction, and determination of folded VEGFR-2 stability. The Hsp90-Pinocembrin complex was found to be quite stable with RMSD value of 0.2nm. Pinocembrin was found to be metabolically stable undergoing phase I metabolism with non-toxic metabolites compared to the standard drug Sorafenib and YLT192., Conclusion: Obtained results propose Pinocembrin as a multi-targeted novel lead compound that bears outstanding antiangiogenic potential against VEGFR-2., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Background: The present study clarifies the molecular interactions of human BACE1 with novel natural ligands and also with the well-known ligand 2, 2, 4-trihydroxychalcone and Galangin for comparison., Objective: The study of enzyme- ligands interaction is interesting, thus description of ligands binding to the active site of target molecule could be beneficial for better understanding the mechanism of the ligand on the target molecule., Methods: Lipinski rule of five and docking study were performed between ligands and enzyme using 'Autodock4.2'., Results: It was found that hydrogen bond interactions play a significant role in the accurate positioning of ligands within the 'active site' of BACE1 to permit docking. Such information may aid to propose the BACE1 -inhibitors and is estimated to aid in the safe medical use of ligands. Selected ligands of BACE1 also inhibit the aggregated amyloid beta peptide. The aggregation of amyloid peptides Aβ1-42 may be responsible for AD., Conclusion: Scope lies in the determination of the 3-dimensional structure of BACE1 and ligands complex by X-ray crystallography to certify the explained data. To validate the enzyme -ligands results, we considered 2, 2, 4-trihydroxychalconeas and Galangin as a positive control. Moreover, the current study verifies that ligands are more capable inhibitors of human BACE1 compared to positive control with reference to ΔG values., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Survivin (IAP proteins) remains an important target for anticancer drug development as it is reported to be over-expressed in tumor cells to enhance resistance to apoptotic stimuli. The study focuses on virtual screening of marine compounds inhibiting survivin, a multifunctional protein, using a computational approach. Structures of compounds were prepared using ChemDraw Ultra 10. Software and converted into its 3D PDB structure and its energy was minimized using Discovery Studio client 2.5. The target protein, survivin was retrieved from RCSB PDB. Lipinski's rule and ADMET toxicity profiling was carried out on marine compounds and the filtered compounds were further promoted for molecular docking analysis and interaction studies using AutoDock Tools 4.0. Molecular docking results revealed that analog (AP 4) of Aplysin, showed very promising inhibitory potential against survivin with a binding energy of -8.75 kcal/mol and Ki 388.28 nM as compared to its known inhibitor, Celecoxib having binding energy of -6.65 kcal/mol and Ki 13.43 μM. AP 4. The analog depicted similarity in pattern when compared to standard. The result proposes AP 4, is an effective molecule exhibiting prominent potential to inhibit survivin and thus promoting apoptosis in tumor cells.

Zika virus (ZIKV) is a mosquito-borne flavivirus distributed all over Africa, South America and Asia. The infection with the virus may cause acute febrile sickness that clinically resembles dengue fever, yet there is no vaccine, no satisfactory treatment, and no means of evaluating the risk of the disease or prognosis in the infected people. In the present study, the efficacy of the host's immune response in reducing the risk of infectious diseases was taken into account to carry out immuno-informatics driven epitope screening strategy of vaccine candidates against ZIKV. In this study, HLA distribution analysis was done to ensure the coverage of the vast majority of the population. Systematic screening of effective dominant immunogens was done with the help of Immune Epitope & ABCPred databases. The outcomes suggested that the predicted epitopes may be protective immunogens with highly conserved sequences and bear potential to induce both protective neutralizing antibodies, T & B cell responses. A total of 25 CD4+ and 16 CD8+ peptides were screened for T-cell mediated immunity. The predicted epitope "TGLDFSDLYYLTMNNKHWLV" was selected as a highly immunogenic epitope for humoral immunity. These peptides were further screened as non-toxic, immunogenic and non-mutated residues of envelop viral protein. The predicted epitope could work as suitable candidate(s) for peptide based vaccine development. Further, experimental validation of these epitopes is warranted to ensure the potential of B- and T-cells stimulation for their efficient use as vaccine candidates, and as diagnostic agents against ZIKV.

4-hydroxy-tetrahydrodipicolinate synthase (DHDPS) is an important enzyme needed for the biosynthesis of lysine and many more key metabolites in Mycobacterium tuberculosis (Mtb). Inhibition of DHDPS is supposed to a promising therapeutic target due to its specific role in sporulation, cross-linking of the peptidiglycan polymers and biosynthesis of amino acids. In this work, a known inhibitor-based similarity search was carried out against a natural products database (Super Natural II) towards identification of more potent phyto-inhibitors. Molecular interaction studies were accomplished using three different tools to understand and establish the participation of active site residues as the key players in stabilizing the binding mode of ligands and target protein. The best phyto-compound deduced on the basis of binding affinity was further used as a template to make similarity scan across the PubChem Compound database (score > = 80 %) to get more divesred leads. In this search 5098 hits were obtained that further reduced to 262 after drug-likeness filtration. These phytochemicallike compounds were docked at the active site of DHDPS.Then, those hits selected from docking analysis that showing stronger binding and forming maximum H-bonds with the active site residues (Thr54, Thr55, Tyr143, Arg148 and Lys171). Finally, we predicted one phytochemical compound (SN00003544), two PubChem-compounds (CID41032023, CID54025334) akin to phytochemical molecule showing better interactions in comaprison of known inhibitors of target protein.These findings might be further useful to gain the structural insight into the designing of novel leads against DapA family.

This study describes in silico validation of the wet lab data from aeroplysinin-1, curcumin and halofuginone using Autodock Tools 4.0. The inhibition patterns of the vascular endothelial cell differentiation and capillary tube formation mediated by anti-angiogenic growth factors from these test compounds were found quite comparable with the in silico results using angiogenic targets (EFGR, bFGF and VEGFR-1). Successful validation of the wet lab results of the selected angiogenic targets by in silico method has led to exploit the hidden potential of in silico tools in preliminary screening of the unknown compounds for anti-angiogenic potential in a cost-effective manner.

Background: Dietary phytochemicals consist of a wide variety of biologically active compounds that are ubiquitous in plants, many of which have been reported to have anti-tumor as well as anti-inflammatory properties., Objective: In the present study, we aimed to validate these findings by using docking protocols and explicate the possible mechanism of action for a dataset of nine phytochemicals namely boswellic acid, 1-caffeoylquinic acid, ellagic acid, emodin, genistein, guggulsterone, quercetin, resveratrol, and sylibinin from different plants against the nuclear factor- kappaB (NF-κB) precursor protein p105, an important transcription factor reported to be overexpressed in breast cancer., Materials and Methods: 2-D structures of all phytochemicals were retrieved from PubChem Compound database and their subsequent conversion into 3-D structures was performed by using online software system CORINA. The X-ray crystallographic structure of the NF-κB precursor p105 was extracted from Brookhaven Protein Data Bank. Molecular docking simulation study was carried out by using AutoDock Tools 4.0., Results: Our results showed significant binding affinity of different phytochemicals with the Rel homology domain of the NF-κB precursor protein p105. Quercetin and 1-caffeoylquinic acid were found to be very effective inhibitors against target molecule as they showed binding energy of -12.11 and -11.50 Kcal/mol, respectively. The order of affinity of other ligands with p105 was found as follows: guggulsterone > sylibinin > emodin > resveratrol > genistein > boswellic acid > ellagic acid., Conclusion: Our in silico study has explored the possible chemopreventive mechanism of these phytochemicals against the NF-κB precursor protein p105 and deciphered that quercetin, 1-caffeoylquinic acid and guggulsterone were the potent inhibitors against target molecule. In addition, large scale preclinical and clinical trials are needed to explore the role of these chemotherapeutic molecules against the NF-κB precursor protein p105 in cure and prevention of breast cancer.

The 2,4,5-triaryl imidazole derivatives (API) were designed, screened and characterized kinetically & thermodynamically against Pepsin and their activity was also tested on the in silico platform. The docking studies of API with Pepsin show that these are novel and unique inhibitors of Aspartic protease. Drug like properties of these compounds were validated in silico based on Lipinski's rule of Five by calculating ClogP, LogS, H-bond acceptors, H-Bond donors, rotational bonds, PSA, PB and BBB values. The Et/Ki and Et/Km values of API show that they follow the Michaelis-Menten kinetics. The binding of inhibitors with proteases was explained by using Van't Hoff plot and thermodynamic parameters viz. free energy (ΔG), Entropy (ΔS) and Enthalpy (ΔH). The Van't Hoff analysis showed that the value of Ki decreases with increase in temperature and the binding of the inhibitor are entropically driven. API act as new potent aspartic protease inhibitors with Ki, for Pepsin, ranges from 3.7 µM to 16.7 µM. Strong hydrophobic groups at C-4 & C-5 position in API favor binding of inhibitors with Pepsin. Experiments also showed that among C-2 aryl substituted imidazole, a 4-substitution on aryl ring is preferred and less polar substituent makes the molecule more active whereas polar substituents at 2-position on C-2 aryl ring makes the molecule less active. The docking studies of API with Pepsin further intensify and validate our results.

Currently used hypolipidemic drugs, Fluvastatin and Atorvastatin, act via inhibiting the rate-limiting enzyme 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase of the mevalonate pathway. The associated severe side-effects of these statins led us to explore the therapeutic potentials of naturally occurring Tocomin (mixture of dietary α-, β-, γ- and δ-tocotrienols). Tocomin (10 mg) was orally administered daily for 10 days before and 12 h after bacterial lipopolysaccharide (200 μg) or 24 h after zymosan (20 mg) or turpentine (0.5 mL) to Syrian hamsters. The data showed that Tocomin significantly reduced the levels of plasma and lipoprotein lipids, cholesterol, apoB, small dense (sd)-LDL as well as LDL in the hyperlipidemia-induced hamsters. Further, the mechanism of action of α-, β-, γ- and δ-tocotrienols was validated by docking studies with HMG-CoA reductase enzyme using the Molegro Virtual Docker. The inhibition of HMG-CoA reductase predicted in terms of MolDockScore and interaction energy suggest the comparative potential in the descending order: Atorvastatin > Fluvastatin ~ δ > γ > β > α. The results favor the daily intake of naturally occurring tocotrienols as dietary supplement in the prevention and treatment of infection/inflammation induced dyslipidemia compared with the hypolipidemic drugs., (Copyright © 2011 John Wiley & Sons, Ltd.)

Background: Tocotrienol (Tocomin) are naturally occurring analogues of vitamin E family and has been reported to possess a potent free radical scavenging activity. In the present study we have initially investigated protective role of tocotrienol against infection and inflammation induced alterations in tissues antioxidant defense system, as well as speculated, via in silico docking studies, that tocotrienol can act by directly binding to antioxidant enzymes., Materials and Methods: Syrian hamsters were injected with bacterial lipopolysaccharide (LPS, 200 microg), zymosan (20 mg), or turpentine (0.5 ml) to mimic acute infection, acute systemic inflammation, and acute localized inflammation, respectively, which are responsible for the generation of plenty of free radicals that causes oxidative stress. Tocomin (10 mg) was administered daily for 10 days before and 12 h after lipopolysaccharides (LPS) or 24 h after turpentine or zymosan injection. Molecular docking studies were performed using Autodock 4.0., Results: Our results show a significant decrease in the activities of antiperoxidative enzymes, glutathione reductase (GR), glutathione peroxidase (GPx), catalase (CAT), superoxide dismutase (SOD), glutathione-s-transferase (GST), as well as reduced glutathione (GSH), in liver and kidney of LPS, turpentine or zymosan stressed hamsters. Feeding of 10 mg Tocomin to stressed hamsters was quite effective in reversing/normalizing the altered levels of enzymatic and nonenzymatic antioxidants in liver and kidney. In order to explore the interaction between tocotrienol and antioxidant enzymes a molecular docking study was performed. The results showed good interaction in term of binding energy and inhibition constant in the following order GR > CAT > SOD > GST > GPx., Conclusion: Our in vivo and in silico results for the first time indicate that tocotrienol significantly alleviate the condition of oxidative stress not only by its potent free radical scavenging properties but also may be by interacting directly and strongly with antioxidant enzymes as proved by molecular docking simulations.

A large variety of ornamental and decorative items are manufactured from bone waste by various unorganized sectors in India. An initial survey indicated that workers were exposed at various phases of final product. The subjects (12 industrial units) were tested for total suspended particulate matter (TSPM), particulate matter <10 microm (PM(10)), and particulate matter <2.5 microm (PM(2.5)). Prevalent levels of TSPM ranged between 2.90 and 5.89 mg m(-3). Respirable fractions of occupational dust as PM(10) and PM(2.5) were found in the range of 0.30-2.08 and 0.26-0.50 mg m(-3), respectively. Cytotoxicity study was conducted using hemolysis as a sensitive marker. In an in vitro study, rat RBCs were exposed to the concentration of 25-1,000 microg/ml for 15-120 min. A considerable variation was observed in the hemolytic activity of samples collected from different areas. At 500 microg/ml concentration, the hemolytic activity (12 h) was found to be in the range of 18-25%. Due to limitation in sample mass of respirable fractions, only one concentration (100 microg/ml/2 h) was used for comparative study on hemolysis of RBCs caused by PM(10) and PM(2.5). Interestingly, the hemolytic activity was more at PM(2.5) than PM(10) and TSPM. These results suggest that the respirable particles are capable of reaching deep into the respiratory system. The finding is significant notably when there are no standards available in occupationally exposed populations. This is the first such study. Data could be of importance to policy makers and regulators.

In this in vitro study we investigated the toxic responses in hepatocytes treated with occupational dust to which workers are exposed in bone-based industrial units. The present study investigated the toxicity mechanism of bone-based occupational dust, from a particular industrial unit, on isolated rat hepatocytes. The hepatocytes were isolated by collagenase perfusion method and cell viability was determined by trypan blue exclusion and MTT [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay treated with occupational dust at 0.1-1.0 mgmL(-1), for 120 min. The cell viability decreased significantly in a concentration-dependent manner. Dust induced significant membrane damage measured by lactate dehydrogenase (LDH) and glutathione (GSH) release in culture media for 30-, 60- and 120 min treatment duration. The toxicity was found to be correlated with the induction of lipid peroxidation (LPO). In addition, nitric oxide (NO), and hydrogen peroxide (H(2)O(2)) generation by occupational dusts were also found to be time- and concentration-dependent. Over all the present study provides initial evidences for the toxic potential of occupational dust generated in bone-based industries and, therefore, the dust exposure to workers in unorganized industrial units should be controlled., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Multidrug resistance (MDR) in microorganisms is a cause of major concern for clinicians and pharmaceutical industries. Continuous development of new antimicrobial drugs with multiple targets and potentials is expected to efficiently combat MDR in these microorganisms. In a continued exploration of new antimicrobial drug leads, 11 marine natural products, semisynthetic, or related synthetic analogs (1-11) and two tobacco cembranoids (12 and 13) were screened for their antimicrobial, antioxidant, and antimutagenic activities. Eight compounds showed varying levels of both antibacterial and antifungal activities. Compounds such as 17-O-methyllatrunculin-A, verongiaquinol, (1S,2E,4R,6R,7E,11E)-2,7,11-cembratriene-4,6-diol), and manzamine-A showed a broad spectrum of activity, inhibiting six of seven tested bacteria with zone of inhibition diameter from 9 to 30 mm. Four of these active compounds also showed antifungal activity. The findings of the in vitro time-kill assay of the most active compound, verongiaquinol, against Staphylococcus aureus indicated its subinhibitory effect at the level lower than the minimal inhibitory concentration (MIC) values (i.e., 2 and 4 µg/mL). At the MIC (8 µg/mL), bacterial cells were completely killed within 18 hours of incubation. DPPH free radical scavenging activity was demonstrated by five compounds in the range of 89.65-36.19% decolorization. Further, four compounds evaluated for their antimutagenic activity against the directly acting mutagens, methyl methanesulfonate and sodium azide, in Salmonella typhimurium strains, interestingly, showed no sign of mutagenicity. Verongiaquinol and manzamine A, in fact, reduced the mutagenicity by 50-75% at a dose of 5 µg/plate in different test strains. Our study seems to provide some novel antimicrobial leads with strong antioxidant potential and the associated ability of antimutagenicity.

The unwarranted connection between diabetes mellitus and breast cancer has gained new ground in recent years. Breast cancer in Saudi females accounts for approximately 21% of all cancers and the prevalence of diabetes mellitus (DM) in Saudi females is also 21.5%. DM is diagnosed in the age group of 30+ years with possible exposure to predisposing factors like hyperinsulinemia and obesity at younger age. Further, 12% of the breast cancer cases are diagnosed in the young females aged 20-34 years. Despite the readily available access to healthcare facilities in the Kingdom, a large number of diabetics, approximately 27.9%, were unaware of having diabetes mellitus. This subpopulation is quite susceptible of developing breast cancer at later age. This review discusses common etiological and predisposing factors for breast cancer and diabetes, regional distribution and possible correlation of diabetes and cancer in Saudi Arabia.

Dimethyl 4,4' -(O-phenylene)bis(3-thioallophanate), commonly known as methyl thiophanate (MT), is a systemic fungicide and suspected carcinogen to humans. In this study, the oxidative potential of this category-III acute toxicant has been ascertained based on its capacity of inducing reactive oxygen species (ROS) and promutagenic 8-oxo-7,8-dihydro-2' -deoxyguanosine (8-oxodG) adducts in DNA. The discernible MT dose-dependent reduction in fluorescence intensity of a cationic dye rhodamine (Rh-123) in human lymphocytes and increased fluorescence intensity of 2',7'-Dichlorodihydro fluorescein diacetate (DCFH-DA) treated cells signifies decreased mitochondrial membrane potential (Delta Psi m) due to intracellular ROS generation. The (32)P-post-labeling assay demonstrated the MT-induced 8-oxodG adduct formation in calf thymus DNA. Thus, it is concluded that MT, as a potent oxidative stressor, produces ROS leading to mitochondrial dysfunction, oxidative DNA damage and mutagenesis.

In the present study, we screened newly synthesized antiviral aminopyrazoloquinoline derivatives for cytotoxic potential in human normal and breast cancer cell lines using apoptosis as biomarker. These derivatives and the well known antiviral drug, acyclovir, were incubated with the normal (MCF-10A, MCF-12A) and cancer (MCF-7, MDA-MB-231) cell lines at 10, 50 and 100 μM for 72 h at 37°C. Both the parent compounds and their sugar derivatives were found to be differentially cytotoxic in various cell lines. MCF-7 cells were more or less completely resistant to all these compounds while MDA-MB-231 cells were significantly killed by apoptosis. The methoxy derivative of aminopyrazoloquinoline (compound 3) was found to be the most cytotoxic in the normal breast epithelial cell lines (MCF-10A and MCF-12A) and MDA-MB-231 cell lines at 100 μM killing over 90% of the cells with up to 80% apoptosis. Interestingly MCF-7 cells showed only up to 50% killing at 100 μM dose with less than 20% apoptosis. Acyclovir did not cause any cytotoxicity, apoptosis or cell cycle arrest in any of the cells lines at the doses tested. Our results suggest that the newly synthesized antiviral compounds have an associated risk of being cytotoxic compared to the acyclovir.

Newly synthesized phthalazine derivatives including copper and platinum complexes were evaluated for cytotoxicity in human breast cancer cell lines. The cells were incubated with the compounds (100 microM) for 72 h and cytotoxicity, apoptosis and DNA content were measured by flow cytometery. Our results suggest that the parent (H1-2), copper (C1-2)- and platinum (P1-2)-derivatized compounds were relatively more active in inducing apoptosis and cell killing in both human breast cancer cell lines, MDA-MB-231 cells being the more sensitive. Other compounds showed weak or no response towards these parameters except H-5 causing 40% apoptosis in MDA-MB-231 cells. Addition of copper or platinum in the structures generally reduced the apoptotic potential. Possible roles for structure activity relationships are discussed.

Several studies have reported the presence of DNA adducts derived from benzo(a)pyrene and other polyaromatics by 32P-postlabeling/TLC by measuring diagonal radioactive zones (DRZs) in lung tissues of human smokers. However, our experimental studies in rodent models, which used modified chromatographic conditions to obtain distinct adduct spots, suggested that cigarette smoke-related lipophilic DNA adducts may not be derived from polycyclic aromatic hydrocarbons (PAHs) or aromatic amines. In the present study, we have performed similar analysis of human lung tissues to study the chemical nature of DNA adducts. Fifty human lung tissues from cancer patients (ages 42-83 years) with active, ex-, or never-smoking status were analyzed for highly lipophilic DNA adducts by nuclease P1- and n-butanol enrichment-mediated 32P-postlabeling assay. All DNA samples yielded low to highly intense adduct DRZs when adducts were resolved by PEI-cellulose TLC in standard high-salt, high-urea solvents. Adduct burden ranged from 6.6 to 2930 per 10(10) nucleotides. However, when adducts were resolved in a different solvent system comprising of high-salt, high-urea in direction 3 and dilute ammonium hydroxide in direction 4, which retained adducts derived from PAHs and aromatic amines on the chromatograms, this yielded no detectable adducts from human lung DNAs. Furthermore, analysis of human lung DNAs mixed with reference adducted DNAs in multisolvent systems confirmed an absence of PAH- and aromatic amine-derived adducts in human smoker lung DNA. To determine the origin of cigarette smoke-associated DNA adducts, calf thymus DNA was incubated with formaldehyde and acetaldehyde, which are known to be present in cigarette smoke in significant quantities. Analysis of purified DNAs by 32P-postlabeling resulted in adduct DRZs in the aldehyde-modified DNAs when adducts were resolved in standard urea-containing solvents, but no adducts were detected when the ammonium hydroxide-based solvent was used, suggesting that even nonpolyaromatic electrophiles can result in adduct DRZs on the chromatograms similar to those from PAH metabolites. Taken together, our data demonstrate that cigarette smoke-associated lung DNA adducts appear on chromatograms as DRZs, consistent with the literature, but they are not related to PAHs and aromatic amines.

This study was conducted to examine the sensitivity of primary skin fibroblasts from Saudi thyroid cancer (TC) patients to ultraviolet (UV) irradiation. Cell survival was studied by a colony forming assay and DNA repair defects with a host cell reactivation (HCR) assay using UV-irradiated Herpes Simplex Virus (HSV). In addition, p53 gene expression was examined in the same TC cells exhibiting enhanced radiosensitivity. Skin fibroblasts from TC patients (n=4) showed significantly enhanced sensitivity to UV radiation. The average UV dose to reduce survival to 37% of the initial survival (D(37)) value (in Jm(-2)) for fibroblasts from TC patients was 4.6 (3.7-5.6) compared to 7.3 (6.3-8.3) for healthy individuals (n=3). UV-sensitive xeroderma pigmentosum (XP) cells, which were used as positive control, were found to be extremely sensitive with a D(37) value of 0.6 Jm(-2). In a host cell reactivation assay, UV-irradiated HSV was tested for its plaque-forming ability (PFA), by plating infected fibroblasts from TC patients (used as host cells) on African Green Monkey (Vero) kidney cells to form plaques. A significant reduction in the PFA of the UV-irradiated virus (about three fold) on TC cells compared to fibroblasts from the healthy subjects was seen, suggesting a DNA-repair deficiency in the primary fibroblasts of the TC patients. Furthermore, no significant accumulation in radiation-induced p53 expression was observed in cells from the TC patients. Our results, based on a relatively small group of subjects, indicate that Saudi TC patients primary fibroblasts (non-cancerous in nature) may be carriers of cancer-susceptible gene(s) arising from defective DNA repair/processing. These results warrant a larger study to investigate the role of UV-induced bulky DNA damage in thyroid cancer susceptibility.

In the past several decades, marine organisms have generously gifted to the pharmaceutical industries numerous naturally bioactive compounds with antiviral, antibacterial, antimalarial, anti-inflammatory, antioxidant, and anticancer potentials. But till date only few anticancer drugs (cytarabine, vidarabine) have been commercially developed from marine compounds while several others are currently in different clinical trials. Majority of these compounds were tested in the tumor xenograft models, however, lack of anticancer potential data in the chemical- and/or oncogene-induced pre-initiation animal carcinogenesis models might have cost some of the marine anticancer compounds an early exit from the clinical trials. This review critically discusses importance of preclinical evaluation, failure of human clinical trials with certain potential anticancer agents, the screening tests used, and choice of biomarkers.

The pro-mutagenic oxidative DNA lesion, 8-oxo-2'-deoxyguanosine (8-oxodG) has been a subject of numerous studies. However, the absolute 8-oxodG levels in tissue DNA reported by various methods have been debated due to its artifactual production during DNA isolation and/or the DNA processing. We have investigated factors that may result in such artifacts during isolation and analysis of DNA as well as means for its prevention. 8-OxodG content was measured by a recently described TLC enrichment-mediated 32P-postlabeling. Liver DNA from 3 month-old, female Sprague-Dawley rats was isolated by a standard solvent-extraction procedure (phenol, phenol:Sevag, and Sevag; 23 degrees C), a modified solvent-extraction procedure (phenol:Sevag, and Sevag; 4 degrees C; KCl-SDS-protein precipitation) or sodium iodide extraction procedure. 8-OxodG was analyzed in the DNA by the 32P-postlabeling assay using a fluorescent light box during the workup, as well as in its absence. The 8-oxodG levels, when the fluorescent light box was used, were in similar range irrespective of the DNA isolation procedure (16.4+/-1.6 to 28.7+/-6 8-oxodG/10(6) nucleotides). However, the values were significantly lower (3.1+/-0.4 to 3.4+/-0.2 8-oxodG/10(6) nucleotides) in the absence of fluorescence light box, room fluorescent light (suspended through the ceiling) and natural room light did not alter the 8-oxodG levels. Further, the addition of 0.3 mM of PBN (N-t-butyl-alpha-phenyl nitrone) or TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl), or 6.8 mM 8-hydroxy-quinoline during the DNA isolation resulted in still lower values (0.8+/-0.1 to 1.8+/-0.5 8-oxodG/10(6) nucleotides) although this reduction was not consistently observed in different experiments. These results suggest that fluorescent light is the major 'culprit' in artifactual production and variability reported in the 8-oxodG levels.

Commercial polychlorinated biphenyls (PCBs) are complete carcinogens in rodents, however, their initiating (DNA damaging) activity has not been conclusively demonstrated. In the present study, we reacted synthetic 2-phenyl-1,4-benzoquinones (BQ) and 2-phenyl-1,4-hydroquinones (HQ) of 2-chloro-, 3-chloro-, 4-chloro-, 3,4-dichloro-, and 3,4,5-trichlorobiphenyls with calf thymus DNA and individual deoxynucleoside-3'-monophosphates for 4 h at 37 degrees C. Analysis of DNA adducts resulting from BQ and HQ derivatives of the test congeners by 32P-postlabeling showed essentially similar adduct patterns. However, the adduct pattern and reactivity differed with the congener used. Quantitatively, 2-chloro-BQ/HQ produced in the highest DNA adduct levels and 3,4,5-chloro-BQ/HQ was the least reactive. Chromatographic comparison of DNA and nucleotide adducts derived from 4-chloro-BQ revealed that cytosine, adenine, and thymidine in the DNA accounted for most of the DNA adducts. Interestingly, none of the adducts in DNA were guanine-derived, even though this mononucleotide was highly reactive. These results suggest that both BQ and HQ derivatives of PCBs are capable of covalently binding to DNA, and chromatographic similarity in adduct patterns resulting from these two metabolites suggest possible involvement of intermediary semiquinone radicals. Experiments are underway to determine their in vivo significance., (Copyright 2002 Elsevier Science B.V.)

There are literally thousands of known agents with potential chemopreventive and antioxidant activity; however, the expanding list of natural and synthetic compounds makes it difficult to test every agent in the widely accepted 2-year animal bioassay and human clinical trials. Therefore, short-term screening assays are needed to sort out the most efficacious compounds for long-term animal studies. In the present study, the identification of chemopreventive agents with efficacious antioxidant potential was explored with a Cu2+-mediated Fenton-type reaction, coupled with oxidative DNA lesion detection by 32P-postlabeling. Several agents inhibited the formation of 8-oxo-2'-deoxyguanosine (8-oxodG), a benchmark oxidative DNA lesion, but ellagic acid, a polyphenol found in berries, offered maximal (>80%) inhibition of 8-oxodG formation. However, a well-known tea polyphenol, epigallocatechin gallate, along with silymarin and D,L-sulforaphane, exhibited a pro-oxidant effect, with 50-70% increase in 8-oxodG induction. In general, our results agree with the reported antioxidant - pro-oxidant activities of the compounds, rendering this in vitro screening assay to be useful in determining the antioxidant potential of compounds rapidly and cost-effectively.

Cigarette smoke contains several oxidants and free radicals. In the present study, we examined the formation of 8-oxo-2'-deoxyguanosine (8-oxodG) in the lungs of female Sprague-Dawley rats exposed to side-stream cigarette smoke for 6 h a day, 7 days a week for 1, 2, 4 and 12 weeks in a whole body-exposure system. The samples were analyzed for 8-oxodG by 32P-postlabeling-TLC enrichment and HPLC-ECD techniques to confirm and compare results. Animals were sacrificed 15 h after the cessation of smoke exposure and lung DNA was isolated by phenol/Sevag extractions in the presence of the free radical traps, 8-hydroxyquinoline (6.8 mM) and N-t-butyl-alpha-phenyl nitrone (500 microM) to minimize artifactual formation of 8-oxodG during sample work up. Analysis of lung DNA by 32P-postlabeling-TLC showed 8-oxodG levels (mean +/- SE) of 1.45+/-0.24, 2.68+/-0.65, 2.23+/-0.28 and 2.93+/-0.54 per 106 nucleotides after 1, 2, 4 and 12 weeks of smoke exposure. The respective values in sham-treated rats were 2.76+/-0.19, 3.69+/-0.20, 1.44+/-0.43 and 2.84+/-0.45 per 106 nucleotides, suggesting no significant effect of smoke exposure on tissue levels of 8-oxodG. HPLC-ECD procedure yielded slightly higher values for 8-oxodG in all groups, however, again significant differences between sham and smoke-exposed groups were not detected. It is concluded that the chronic exposure to side-stream cigarette smoke does not enhance the formation of 8-oxodG in rat lungs.

To use oxidative DNA lesions as biomarkers of exposure and disease in the human scenario, the assay should not only be sensitive but also applicable to small quantities of DNA that is obtained from human tissue biopsies. Previously, we reported a nonenrichment (32)P-postlabeling method for measuring the oxidative DNA lesion, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) [Devanaboyina, U., and Gupta, R. C. (1996) Carcinogenesis 17, 917-924]. Here we report a substantial improvement of the assay by enriching 8-oxo-dGp by TLC prior to (32)P-labeling. Thus, unmodified nucleotides were removed by 1-directional polyethyleneimine (PEI)-cellulose TLC in 0.2 M formic acid. 8-Oxo-dGp, retained close to the origin, was eluted in 2 M triethylammonium acetate (pH 7.0), lyophilized, (32)P-labeled, resolved by TLC, and quantitated. The 8-oxo-dG signal was found to increase linearly with increasing amount of DNA. Its recovery was found to be 55-70%, as determined by using synthetic 8-oxo-dGp. Since the modified assay allowed using larger quantities of DNA and higher specific activity [gamma-(32)P]ATP than those used in the nonenrichment procedure, the signal-to-noise ratio increased to 30-100:1 from 3-5:1. The assay has a sensitivity of detection of <1 8-oxo-dG/10(7) nucleotides using submicrogram to microgram DNA. The 8-oxo-dG levels (mean +/- SE; n = 5) in the liver, lung, heart, bladder, and trachea DNA of 3 month-old female Sprague-Dawley rats were found to be 1.05 +/- 0.24, 0.97 +/- 0.09, 0.75 +/- 0.15, 0.79 +/- 0.15, and 1.17 +/- 0.28 per 10(6) nucleotides, respectively.

Dithiolethiones are currently one of the most promising classes of cancer chemopreventive agents that exhibit antitumorigenic properties at numerous organ sites against several classes of carcinogens. In the current study, we examined the effects of 2 dithiolethiones, 1,2-dithiole-3-thione (D3T) and its structural analogue oltipraz, on DNA adduction induced by the potent mammary carcinogen dibenzo-[a,l]pyrene (DBP) in vivo. Female Sprague-Dawley rats were provided dietary D3T and oltipraz (500 ppm each) for I week followed by a single intragastric dose of DBP (8 micromol/kg body weight) and killed 5 days later. D3T inhibited DBP-DNA adduction from 78% to 82% in all tissues examined, while oltipraz was equally effective in the lung and liver but less effective in the mammary glands, inhibiting DBP-DNA adduction by nearly 60%. These data coupled with their broad anti-tumor specificity support the use of D3T and oltipraz as cancer-preventive agents in clinical trials.

Indole-3-carbinol (I3C) found in various cruciferous vegetables has been shown to exert anti-carcinogenic activity in several target organs. In this study, we have investigated the effects of I3C on cigarette smoke-related lipophilic DNA adduct formation, potentially a key step in chemical carcinogenesis. Female Sprague-Dawley rats were exposed to sidestream cigarette smoke in a whole-body exposure chamber for 6 h per day, 7 days a week for 4 weeks. Control animals received only vehicle while the intervention groups received I3C (1. 36 or 3.40 mmol/kg, b.wt.) daily by gavage starting from 1 week prior to smoke initiation until the end of the experiment. Analysis of tissue DNA by nuclease P1-mediated 32P-postlabeling showed one major and several minor smoke-related adducts in lung, trachea, heart and bladder. The high dose of I3C significantly inhibited the major adducts in lung (#5) and trachea (#3) by 55% each; minor adducts were slightly inhibited (20-40%). The low dose of I3C showed lesser degree of inhibition (30-40%) in both lung and trachea; however, it was found statistically significant in lung only. The major smoke-related adduct in bladder (#2) was strongly inhibited (>65%) by high dose of I3C approaching adduct levels achieved in sham-exposed rats. A small but statistically significant decrease in the smoke-related DNA adduct (#5) in heart tissue was also observed by intervention with high dose I3C. Low levels (30-50 adducts/10(10) nucleotides) of I3C-derived DNA adducts were also found in all the tissues examined although their significance remains unknown. These data show significant inhibition of cigarette smoke-related DNA adducts by I3C, particularly in the lung, trachea, and bladder.

Dibenzo[a,l]pyrene (DBP), an environmentally significant polycyclic aromatic hydrocarbon (PAH), is one of the most potent carcinogens with greater carcinogenicity in rodent mammary glands and skin than 7,12-dimethylbenz[a]anthracene or benzo[a]pyrene, respectively. In this study, we have examined the formation and persistence of stable DNA adducts in rats administered a carcinogenic intramammillary (i.m.) dose of DBP (0.25 micromol/gland). 32P-post-labeling analysis of mammary epithelial DNA 6 h, and 2, 5 and 14 days post-treatment produced one major (approximately 30%) and at least six minor adducts. Non-target tissue DNA (lung, heart, bladder and pancreas) also showed essentially the same adduct pattern as did mammary DNA, except liver which resulted in four additional adduct spots. The mammary DNA was most adducted (2640 +/- 532 adducts/10(9) nucleotides) on day 5 while the other tissue DNAs had 10- to 65-fold lower adduct levels (lung > liver > heart > bladder > pancreas). Adduct levels continued to increase at all time points examined for all tissues, except mammary tissue which showed a decline ( approximately 40%) on day 14. Chromatographic comparison with adducts formed in vitro by reaction of syn- and anti-DBP-11, 12-diol-13,14-epoxides (DBPDEs) with DNA and individual nucleotides indicated that the in vivo adducts were deoxyadenosine- and deoxyguanosine-derived, formed by interaction with both the anti- and syn-isomers; the adenine-derived adducts comprised 60-75% of the total adducts. However, the liver-specific DNA adducts (nos 8-11) were not derived from any of the DBPDE isomers. Our data show: (i) significantly higher DBP-DNA adduction in mammary tissue as compared with non-target tissues, which is consistent with its mammary carcinogenicity; (ii) adenine is highly reactive towards DBP metabolites as has been observed for many other PAHs; and (iii) the peak binding of DBP with DNA was shifted beyond 14 days for the non-target tissues by i.m. route of exposure.

A significant depletion in the content of glutathione (GSH) and alteration in GSH redox system enzymes were observed in the lung of chrysotile-exposed animals (5 mg) during different developmental stages of asbestosis. In the alveolar macrophages (AM) of exposed animals, the depletion in GSH started from day 1 and reached a maximum at day 16, whereas in lung tissue the maximum depletion was observed when fibrosis has matured. It appears that cellular GSH depletion triggers oxidative stress in the system as observed from increased thiobarbituric acid reactive substance (TBARS) production and alteration in the activities of glutathione peroxidase (GPx), glutathione reductase (GR), glucose 6-phosphate dehydrogenase (G6PD) and glutathione S-transferase (GST), the enzymes regulating oxidative tone. The depletion in GSH was also observed in red blood cells (RBC) of the exposed animals reaching a maximum when fibrosis matured. Thus the observed depletion in GSH, ascorbic acid and alteration in GSH redox system enzymes may be involved in fibrosis and carcinogenesis induced by chrysotile.

Bay-region diol epoxides are considered the putative ultimate carcinogens of polynuclear aromatic hydrocarbons. However, the results of studies on tumorigenesis and DNA binding of benzo[a]pyrene (BP) and its bay-region diol epoxide, (+)-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyren e [(+)-anti-BPDE] suggest that, in addition to anti-BPDE, other reactive metabolite(s) of BP may also be involved in BP-induced carcinogenesis. Recent studies have demonstrated that 3-hydroxy-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a ]pyrene (anti-BPTE) is another highly reactive metabolite of BP. In order to identify syn- and anti-BPTE-derived DNA adducts and their base selectivity, we synthesized both compounds by two different methods and reacted in vitro with calf thymus DNA and individual nucleotides. The resultant adducts were analyzed by nuclease P1-enhanced 32P-postlabeling. Anti-BPTE produced three major and several minor adducts with DNA; dAp and dGp were the preferred substrates, while dCp and dTp were the least reactive. In contrast, syn-BPTE produced two major adducts each with DNA and dGp; dAp generated only one adduct. Co-chromatography of anti-BPTE-derived DNA adducts with those of mononucleotide adducts revealed that the major adducts in DNA were guanine derived. Further, co-chromatographic results revealed that the anti-BPTE-DNA adducts were distinctly different from that of anti-BPDE-DNA adducts. These observations indicate that both syn- and anti-BPTE can react with DNA bases and these DNA adducts may also contribute to BP-induced carcinogenesis.

Exposure to tobacco smoke has been implicated in the increased incidence of cancer and cardiovascular diseases. This report describes various experimental studies in animals that were carried out to determine the ability of cigarette smoke to form DNA adducts and to define chromatographic nature of the major adducts. Tissues from rodents exposed to mainstream or sidestream cigarette smoke in nose-only and whole-body exposure systems, respectively, for different durations were analyzed for DNA adducts by 32P-postlabeling assay. The results showed essentially similar qualitative patterns in various respiratory (lung, trachea, larynx) and non-respiratory (heart, bladder) tissues of smoke-exposed rats. However, adduct pattern in the nasal mucosa was different. The mean total DNA adducts in various tissues expressed as per 1010 nucleotides exhibited the following order: heart (700)>lung (420)>trachea (170)>larynx (150)>bladder (50). Some qualitatively identical adducts were routinely detected in tissues from sham-treated rats but at greatly reduced levels (5- to 25-fold). The levels of lung DNA adducts increased with the duration of exposure up to 23 weeks and returned to control levels 19 weeks after the cessation of exposure. Species-related differences in adduct magnitude and patterns were observed among rats, mice and guinea pigs; mouse being the most sensitive to DNA damage and guinea pig the least sensitive. Whole-body exposure of rats to sidestream cigarette smoke also enhanced the pre-existing DNA adducts by several fold in different tissues. Selective chromatography, and extractability in butanol suggested lipophilic nature of smoke-associated DNA adducts, which were, however, recovered significantly better in nuclease P1 than butanol enrichment procedure. The major smoke-associated adducts were chromatographically different from any of the reference adducts of polycyclic aromatic hydrocarbons (PAHs) co-chromatographed with the smoke DNA samples. Because PAH-DNA adducts are recovered with equal efficiency by the two enrichment procedures, the above observations suggested that smoke-associated adducts are not related to typical PAHs, like benzo[a]pyrene. It is concluded that cigarette smoke increased the levels of pre-existing endogenous DNA adducts (the so-called I-compounds) in animal models and that these adducts are unrelated to those formed by typical PAHs., (Copyright 1999 Elsevier Science B.V.)

The present study investigated the effects of dietary oltipraz on cigarette smoke-related lipophilic DNA adduct formation. Female Sprague-Dawley rats were exposed daily to sidestream cigarette smoke in a whole-body exposure chamber 6 h/day for 4 consecutive weeks. One group of rats was maintained on control diet while another group received the same diet supplemented with either a low (167 p.p.m.) or high (500 p.p.m.) dose of oltipraz, starting 1 week prior to initiation of smoke exposure until the end of the experiment. Analysis of lipophilic DNA adducts by the nuclease P1-mediated 32P-post-labeling showed up to five smoke-related adducts. Adduct no. 5 predominated in both the lung and the heart while adduct nos 3 and 2 predominated in the trachea and bladder, respectively. Quantitative analysis revealed that the total adduct level was the highest in lungs (270+/-68 adducts/10(10) nucleotides), followed by trachea (196+/-48 adducts/10(10) nucleotides), heart (141+/-22 adducts/10(10) nucleotides) and bladder (85+/-16 adducts/10(10) nucleotides). High dose oltipraz treatment reduced the adduct levels in lungs and bladder by >60%, while the reduction in lungs in the low-dose group was approximately 35%. In trachea, the effect of low and high dietary oltipraz on smoke DNA adduction was equivocal, while smoke-related DNA adducts in the heart were minimally inhibited by high-dose oltipraz. In a repeat experiment that employed a 3-fold lower dose of cigarette smoke, oltipraz (500 p.p.m.) was found to inhibit the formation of DNA adducts in rat lungs and trachea by 80 and 65%, respectively. These data clearly demonstrate a high efficacy of oltipraz in inhibiting the formation of cigarette smoke-induced DNA adducts in the target tissues.

Due to the large and expanding number of potential cancer chemopreventive agents, there is an increasing need for short term tests to study the efficacy and mechanisms of these agents. In this study, we have employed a microsome-mediated test system to study the effect of several suspected chemopreventive agents on the DNA adduct formation capacity of the potent mammary carcinogen, dibenzo[a,l]pyrene (DBP). Bioactivation of DBP by Aroclor 1254-induced rat liver microsomes in the presence of calf thymus DNA (300 microg/ml) resulted in the formation of one major and six other prominent DNA adducts (324 adducts/10(7) nucleotides). These adducts were previously determined to be deoxyadenosine (dA) and deoxyanosine (dG)-derivatives of both anti- and syn-DBP-11,12-diol-13,14-epoxides (DBPDE). Intervention with ellagic acid, chlorophyllin, benzyl isocyanate (BIC), oltipraz or genistein (150 microM) strongly diminished DBP-DNA adduction by > or = 75%. Linoleic acid, curcumin and butylated hydroxytoluene (BHT) also significantly inhibited DBP DNA adduction (26-46%) while N-acetylcysteine (NAC) had no effect. Moreover, nonenzymatic studies with anti- and syn-DBPDE isomers revealed that chlorophyllin, ellagic acid, BIC and BHT may be inhibiting DBP-DNA adduction in an enzymatic-independent manner since these agents diminished DBPDE-DNA adduction by 30-75%. Genistein, oltipraz and curcumin did not diminish DBPDE-DNA adduction and therefore most likely require the presence of the microsomal subcellular fraction to inhibit DBP-DNA adduction.

Dibenzo[a,l]pyrene (DBP) is one of the most potent bacterial mutagen and mammary carcinogens. When DBP (50 microM) was incubated with calf thymus DNA (300 microg/ml) in the presence of liver microsomes from beta-naphthoflavone (beta-NF)- or Aroclor 1254-treated rats, at least eight adduct spots were detected as analyzed by nuclease P1-enhanced 32P-postlabeling assay. DNA adduction was enhanced by nearly 20- and 60-fold with beta-NF- and Aroclor 1254-induced microsomes, respectively, as compared with uninduced microsomes, suggesting a possible involvement of CYP1A family in DBP activation. Inclusion of the selective P4501A1 inhibitor, alpha-naphthoflavone (50 microM) in the activation reaction almost completely (>98%) abolished adduct formation further supporting involvement of P4501A in DBP activation. Analysis of DNA and 2'-deoxynucleosides 3'-mononucleotide reacted with anti- and syn-DBP-11,12-diol-13,14-epoxides (DBPDEs) and co-chromatography analyses in multiple solvents showed that the microsomal DBP-DNA adducts were derived by interaction of both anti- and syn-DBPDEs with adenine and guanine in DNA in the following order: anti-DBPDE-dA approximately syn-DBPDE-dG >> anti-DBPDE-dG approximately syn-DBPDE-dA. It is concluded that (i) most or all DBP adducts were P4501A-mediated; (ii) both the anti- and syn-stereoisomers were involved in the DNA adduct formation; and (iii) both adenine and guanine in the DNA contributed equally to the formation of the major and minor adducts.

Several observations, including studies from this laboratory, demonstrate that asbestos generates free radicals in the biological system that may play a role in the manifestation of asbestos-related cytotoxicity and carcinogenicity. It has also been demonstrated that iron associated with asbestos plays an important role in the asbestos-mediated generation of reactive oxygen species. Exposure to asbestos leads to degradation of heme proteins such as cytochrome P450-releasing heme in cytosol. Our simulation experiments in the presence of heme show that such asbestos-released heme may increase lipid peroxidation and can cause DNA damage. Further, heme and horseradish peroxidase (HRP) can cause extensive DNA damage in the presence of asbestos and hydrogen peroxide/organic peroxide/hydroperoxides. HRP catalyzes oxidation reactions in a manner similar to that of prostaglandin H synthetase. Iron released from asbestos is only partially responsible for DNA damage. However, our studies indicate that DNA damage mediated by asbestos in vivo may be caused by a combination of effects such as the release and participation of iron, heme, and heme moiety of prostaglandin H synthetase in free radical generation from peroxides and hydroperoxides.

Dibenzo[a,l]pyrene (DBP) has recently emerged as a potent environmental carcinogen having greater carcinogenicity in the rat mammary epithelial glands than 7,12-dimethylbenz[a]anthracene (DMBA), previously considered to be the most potent mammary carcinogen and benzo[a]pyrene (BP), a ubiquitous environmental carcinogen. Previous studies on the tumor-initiating potential of DBP, DMBA, and BP demonstrated that DBP was 2.5 times more potent in inducing the tumors in mouse skin and rat mammary glands than DMBA; BP was a weak mammary carcinogen in these animals. The present study was designed to investigate if the significantly increased mammary carcinogenicity of DBP over DMBA and BP was related to increased DNA adduction at the target site. Female Sprague-Dawley rats were treated by intramammillary injection with an equimolar dose of 0.25 micromol/gland of DBP, DMBA, and BP at the 3rd, 4th and 5th mammary glands on both sides. 32P-Postlabeling analysis of mammary epithelial DNA of rats treated with DBP produced two major (nos. 3 and 6) and at least 5 minor adducts. DMBA treatment resulted in one major and 4 minor DNA adducts while BP produced one major and two minor adducts. Quantitation of the adduct radioactivity revealed that DNA adduction was 6- and 9-fold greater in DBP-treated animals than in BP- and DMBA-treated animals, respectively. The adduct levels per 10(9) nucleotides in mammary epithelial cells for DBP, BP and DMBA were in the following descending order: 1828 +/- 378, 300 +/- 45 and 207 +/- 72, respectively. Tissue distribution of DNA adducts in non-target organs following DBP treatment showed similar adduct pattern as found in the mammary epithelial cells except the liver, which resulted in 4 additional adduct spots; vehicle-treated tissue DNA processed in parallel did not show any detectable adducts. DMBA- and BP-DNA adduct patterns in various tissues were similar to that found in mammary epithelial cells, however, significant quantitative differences were found; BP-DNA adducts were undetectable in the pancreas and bladder. Quantitation of adduct radioactivity showed a 15- to 60-fold lower DBP-DNA adduction in these tissues than the levels found in the mammary tissue; similarly 5-20 and 30-100 times lower DNA adduction was found following treatment with DMBA and BP, respectively. The significantly increased binding of DBP to the mammary epithelial DNA over BP and DMBA is in concordance with its known higher mutagenicity and tumorigenicity.

In order to examine the pulmonary toxicity of kerosene oil and its combustion product (soot) in asbestos-exposed rats, various biochemical and chemical parameters were assayed. Treatment of rats with a single intratracheal dose of chrysotile asbestos (5 mg) and kerosene (50 microliters) or its soot (5 mg) in combination led to an increased number of pulmonary alveolar macrophages (PAM), elevated levels of hydrogen peroxide, and thiobarbituric acid-reacting substances, alterations in the activities of primary (glutathione peroxidase and catalase) and secondary (glutathione reductase and glucose-6-phosphate dehydrogenase) endogenous antioxidant enzymes, and depletion in the levels of glutathione in PAM compared to the chrysotile, kerosene, or soot alone. These changes may indicate the generation of oxidative stress in the macrophages. The resulting oxidative stress may be subsequently critical in collapsing the cellular membrane, which may change the cell membrane permeability and may also damage the phagolysosomal membrane, thereby releasing the membrane bound enzymes as indicated by an increased leakage of intracellular acid phosphatase and lactate dehydrogenase. The injury to macrophages may trigger events that lead to lung fibrosis and/or malignancies in the exposed animals. This study may be helpful in understanding the etiology of certain clinical and pathological disorders in the population exposed simultaneously to both asbestos and kerosene or its combustion products.

The glutathione and vitamin E dependent protection of lipid peroxidation in an NADPH (0.4 mM) and chrysotile (500 micrograms/ml) containing system were investigated in vitro in rat lung microsomes. Addition of 1 mM glutathione to the above reaction system containing microsomes supplemented with vitamin E (1 nmol/mg protein) reduced lipid peroxidation. Similar protection by glutathione could be observed in normal unsupplemented microsomes though the degree of protection was less pronounced. Addition of free radical scavengers such as, superoxide dismutase (100 units/ml), catalase (150 units/ml), mannitol (1 mM) and beta-carotene (0.5 mM) to the reaction system showed an insignificant effect on lipid peroxidation. When the reaction was carried out in absence of glutathione, vitamin E content of peroxidizing microsomes decreased rapidly. In this system a concomitant increase in the activity of microsomal glutathione-S-transferase was observed which may serve as an alternative pathway to detoxify lipid peroxides. Addition of glutathione alone to the reaction system prevented both against the loss in vitamin E content and increase in the activity of glutathione-S-transferase. Supplementation of both vitamin E and glutathione was found to be effective in lowering glutathione-S-transferase activity to that of normal basal level. Our results suggest that chrysotile-mediated stimulation of NADPH-dependent lipid peroxidation may be due to hampering of glutathione-dependent protection which may ultimately exhaust membrane bound vitamin E. Our data further suggest that the lung tissue may have an inbuilt mechanism whereby glutathione-S-transferase may be triggered to cope with the excessive production of lipid peroxides.

Our previous studies suggested that body fluids such as serum can serve as a novel surrogate for DNA-containing tissues to overcome the major limiting factor of tissue availability in human biomonitoring assessments. We have now examined the detectability of DNA-reactive metabolites (DRMs) in the serum and tissues of male C57BL/6 mice administered up to 10 i.p. injections on alternate days of individual polynuclear aromatic hydrocarbons (PAHs) or a mixture containing 0.01, 0.1, 1.0 and 5.0 mg/kg body wt each of benzo[a]pyrene (BP), cyclopenta[cd]pyrene (CPP) and benzo[k]fluoranthene (BF) in 200 microl sunflower oil. Four hours after the last dose, blood serum was separated and incubated with salmon testes DNA (st-DNA). 32P-Postlabeling of the st-DNA showed essentially the same adduct pattern as found in the tissue DNA, indicating that DRMs of the PAHs used were presumably sequestered and accumulated in the serum proteins. Serum DRMs were detectable at all doses tested following exposure to a mixture of BP, CPP and BF, with varying degrees of detection of the individual PAHs. At doses > or = 1 mg/kg, CPP was more potent than BP and BF in producing serum DRMs as well as tissue DNA adducts. At lower doses, however, BP was more potent. Serum DRMs were enhanced 7-20 times when the animals received 1 mg/kg each of these PAHs in the form of a mixture as compared with individual PAHs, however, tissue DNA adduction was enhanced to a lesser degree (1.5- to 2-fold). Results from these studies suggest that: (i) DNA adduction by a particular carcinogen can be significantly higher when administered in the form of a mixture than administered individually; (ii) DNA-reactive metabolites can be detected by 32P-postlabeling in the serum of animals treated with environmentally significant doses of an artificial carcinogen mixture. This approach has implications for human biomonitoring and offers an advantage because this obviates the need to acquire human tissue DNA, a major obstacle in population biomonitoring studies.