Identifying the alpha-glucosidase inhibitory potential of dietary phytochemicals against diabetes mellitus type 2 via molecular interactions and dynamics simulation.

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.

Protective effect of dietary tocotrienols against infection and inflammation-induced hyperlipidemia: an in vivo and in silico study.

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-, ß-, 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-, ß-, 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 > ß > 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.

Antimicrobial, antioxidant, and antimutagenic activities of selected marine natural products and tobacco cembranoids.

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.

Genotoxic fungicide methyl thiophanate as an oxidative stressor inducing 8-oxo-7,8-dihydro-2' -deoxyguanosine adducts in DNA and mutagenesis.

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.

Molecular Mechanism of Cancer Susceptibility Associated with Fok1 Single Nucleotide Polymorphism of VDR in Relation to Breast Cancer

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 case­control 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.

Structural Insight into the Mechanism of Dibenzo[a,l]pyrene and Benzo[a]pyrene-Mediated Cell Proliferation Using Molecular Docking Simulations.

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.

Evaluation and Elucidation Studies of Natural Aglycones for Anticancer Potential using Apoptosis-Related Markers: An In silico Study.

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-3ß-ol), solasodine (Solasod-5-en-3ß-ol), and tomatidine (5α-Tomatidan-3ß-ol)] induce apoptosis by inhibiting the p53-MDM2 complex, p21Waf1/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.

Development of In Silico Protocols to Predict Structural Insights into the Metabolic Activation Pathways of Xenobiotics.

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.

Molecular Interaction and Computational Analytical Studies of Pinocembrin for its Antiangiogenic Potential Targeting VEGFR-2: A Persuader of Metastasis.

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.

Marine Drugs: A Hidden Wealth and a New Epoch for Cancer Management.

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.

Molecular docking analysis of aplysin analogs targeting survivin protein.

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.

Screening and Elucidation of Selected Natural Compounds for Anti- Alzheimer's Potential Targeting BACE-1 Enzyme: A Case Computational Study.

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 Aß1-42 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.

In silico CD4+, CD8+ T-cell and B-cell immunity associated immunogenic epitope prediction and HLA distribution analysis of Zika virus.

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.

Evaluation of apoptosis-induction by newly synthesized phthalazine derivatives in breast cancer cell lines.

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.

Identification of potential leads against 4-hydroxytetrahydrodipicolinate synthase from Mycobacterium tuberculosis.

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.

A rapid screening assay for antioxidant potential of natural and synthetic agents in vitro.

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.

Novel Marine Compounds: Anticancer or Genotoxic?

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.

Artifactual formation of 8-oxo-2'-deoxyguanosine: role of fluorescent light and inhibitors.

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.

Interaction of benzoquinone- and hydroquinone-derivatives of lower chlorinated biphenyls with DNA and nucleotides in vitro.

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.