Antimutagenic and antitumorigenic activities of nordihydroguaiaretic acid.

Nordihydroguaiaretic acid (NDGA), which occurs in the resinous exudates of many plants is used as an antioxidant in fats and oils. In this study we show that NDGA inhibited the mutagenicity of methyl methanesulfonate, benzo[a]pyrene (BP), 2-aminofluorene, and aflatoxin B1 in Salmonella typhimurium strain TA100 or TA98 in the absence and presence of rat hepatic microsomal activation system. The addition of NDGA during and after nitrosation of methylurea (MU) resulted in a dose-dependent inhibition of mutagenicity induced by nitrosation products of MU. In a two-stage skin tumorigenesis protocol using 7,12-dimethylbenz[a]anthracene (DMBA) as the initiating agent followed by twice weekly applications of 12-O-tetradecanoylphorbol-13-acetate (TPA) as tumor promoter, pretreatment of animals with NDGA prior to DMBA application, afforded significant protection against skin tumorigenicity in female SENCAR mice. In additional studies, skin application of NDGA also inhibited the binding of topically applied [3H]BP and [3H]DMBA to epidermal DNA. When assessed in the anti-tumor promotion protocol, pretreatment of animals with NDGA before each application of TPA in DMBA-initiated mouse skin, resulted in 72% decrease in the total number of tumors when compared to non-NDGA pretreated animals. The possible mechanism(s) of the antimutagenic and anti-tumorigenic activities may be due to the multiple effects of NDGA as inhibitor of the carcinogen metabolism and DNA-adduct formation, scavenger of carcinogen free radicals, and as inhibitor of TPA-induced ornithine decarboxylase activity.     

In situ evidence for the involvement of superoxide anions in cutaneous porphyrin photosensitization

Dihematoporphyrin ether, also known as Photofrin-II (Pf-II) is currently used in the diagnosis and management of a variety of epithelial neoplasms, in a modality known as photodynamic therapy (PDT). A major drawback of these porphyrins for PDT is their ability to evoke prolonged cutaneous photosensitization. The mechanism of tumor ablation and cutaneous photosensitization by these photosensitizers is thought to relate to the generation of one or more reactive oxygen species such as superoxide anion, singlet oxygen and hydroxyl radical. However, the role of these oxygen species has not been established unequivocally. In this study, the mechanism of Pf-II-mediated cutaneous photosensitization was examined using murine ear swelling as a marker. The mice treated with Pf-II and light demonstrated two-fold enhancement of ear swelling whereas animals treated with the SOD mimic, beta-carotene and dimethyl sulfoxide (DMSO) had considerably less ear swelling (p less than 0.01). The observed protective effect was dependent on the dose of each quencher and followed the pattern SOD mimic DMSO beta-carotene. The histopathologic alterations caused by Pf-II photosensitization were significantly alleviated by pretreatment with SOD mimic whereas beta-carotene and (DMSO) were less effective. Inhibitors of superoxide dismutase (sodium diethyldithiocarbamate) and catalase (hydroxyl amine and 3, amino 1,2,4-triazole) augmented Pf-II-mediated cutaneous photosensitization. These data provide the first in vivo evidence for the involvement of superoxide anion in cutaneous porphyrin photosensitization.     

Inhibition of epidermal metabolism and DNA-binding of benzo[a]pyrene by ellagic acid

Ellagic acid, a common plant phenol, was shown to be a potent inhibitor of epidermal microsomal aryl hydrocarbon hydroxylase (AHH) activity in vitro, and of benzo[a]pyrene (BP)-binding to both calf thymus DNA in vitro and to epidermal DNA in vivo. The in vitro addition of ellagic acid (0.25-2.0 microM) resulted in a dose-dependent inhibition of AHH activity in epidermal microsomes prepared from control or carcinogen-treated animals. The I50 of ellagic acid for epidermal AHH was 1.0 microM making it the most potent inhibitor of epidermal AHH yet identified. In vitro addition of ellagic acid to microsomal suspensions prepared from control or coal tar-treated animals resulted in 90% inhibition of BP-binding to calf thymus DNA. Application of ellagic acid to the skin (0.5-10.0 mumol/10 gm body wt) caused a dose-dependent inhibition of BP-binding to epidermal DNA. Our results suggest that phenolic compounds such as ellagic acid may prove useful in modulating the risk of cutaneous cancer from environmental chemicals.     

Cutaneous metabolism of benzo[a]pyrene: comparative studies in C57BL/6N and DBA/2N mice and neonatal Sprague--Dawley rats

The metabolism of the polycyclic aromatic hydrocarbon (PAH) carcinogen benzo[a]pyrene (BaP) was studied using microsomes prepared from the skin of the mouse and rat. Topical application of the polychlorinated biphenyl (PCB) Aroclor 1254 or the PAH 3-methylcholanthrene (3-MC) to the skin of the C57BL/6N and DBA/2N mouse and the Sprague-Dawley rat caused statistically significant enhancement of cutaneous microsomal aryl hydrocarbon hydroxylase (AHH) activity in each animal. PCB was a more potent inducer of the enzyme than was 3-MC. BaP metabolism by skin microsomes from the same animals was assessed using high performance liquid chromatography (HPLC). The skin of untreated animals metabolized BaP into 9,10-, 7,8- and 4,5-dihydrodiols, phenols and quinones. Skin application of PCB caused greater than 16–18-fold enhancement of BaP metabolism in the C57BL/6N mouse and the rat and 2–5-fold enhancement in the DBA/2N mouse. Skin application of 3-MC enhanced BaP metabolism 2–8-fold in the C57BL/6N mouse and 5–10-fold in the rat and had no effect in the DBA/2N mouse. The formation of procarcinogenic metabolite BaP-7, 8-diol was greatly enhanced (4–12-fold) by treatment with the PCB and 3-MC in the tumor susceptible C57BL/6N mouse and in the tumor-resistant neonatal Sprague-Dawley rat. In contrast, the formation of BaP-7,8-diol was either slightly enhanced (2-fold) or unaffected by treatment with the PCB or 3-MC in the tumor-resistant DBA/2N mouse. Our data indicate that neither the patterns of metabolism nor the amount of BaP-7,8-diol formation in the skin are reliable predictors of tumor susceptibility to the PAH in rodent skin.     

Hepatic and extra-hepatic glutathione-S-transferase activity in wild pigeons (Columba livia)

Glutathione-S-transferase (EC 2.5.1.18) activity was assayed in hepatic and extra-hepatic tissues of pigeons using l-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene as substrates. Gluthathione-S-transferase activity towards 1-chloro-2,4-dinitrobenzene in pigeon was in the order: kidney > liver > testes > brain > lung> heart. The enzyme activity with 1-chloro-2,4-dinitrobenzene as substrate was 40–44 times higher in pigeon liver and kidney than that observed with 1,2-dichloro-4-dinitrobenzene as substrate.K _m values of hepatic and renal glutathione transferase with l-chloro-2,4-dinitrobenzene as substrate were 2.5 and 3 mM respectively. Double reciprocal plots with varying reduced gluthathione concentrations resulted in biphasic curves with twoK _m values (liver 0.31 mM and 4mM; kidney 0.36 mM and 1.3 mM). The enzyme activity was inhibited by oxidized gluthathione in a dose-dependent pattern. 3-Methylcholanthrene elicited about 50% induction of hepatic glutathione transferase activity whereas phénobarbital was ineffective.     

Thyroid hormone-induced changes in the hepatic monooxygenase system, heme oxygenase activity and epoxide hydrolase activity in adult male, female and immature rats

In 8-day-old rat pups, pretreatment with a single injection of L-triiodothyronine or L-thyroxine decreased hepatic cytochrome P-450 content, aminopyrine N-demethylase activity and epoxide hydrolase activity but increased hepatic microsomal cytochrome c reductase, 7-ethoxyresorufin O-deethylase and heme oxygenase activities without significantly altering UDP-glucuronosyltransferase activity (towards o-aminophenol) or the microsomal yield.

In adult rats of either sex such single injections of L-triiodothyronine failed to significantly alter these enzyme activities. However, multiple injections evoked changes similar to those observed in the pups, in all these enzyme activities, except that 7-ethoxyresorufin O-deethylase activity was slightly decreased rather than increased.

These findings demonstrate that: (1) The hepatic monooxygenase system in the rat pup is more responsive to thyroid hormones than that in adult. (2) Thyroid hormones can decrease rat liver cytochrome P-450 content and its dependent monooxygenase activity independently of sexual maturity. (3) Thyroid hormones also decrease hepatic epoxide hydrolase activity in both pups and adults. Thus, hyperthyroidism could render the rat pup more susceptible to hepatotoxicity from electrophilic epoxides which utilize microsomal epoxide hydrolase as the major detoxication pathway.     

Evidence for the involvement of singlet oxygen in the photodestruction by chloroaluminum phthalocyanine tetrasulfonate

In recent years, choloroaluminum phthalocyanine tetrasulfonate (A1PCTS) has been shown to be a promising photosensitizer for the photodynamic therapy (PDT) of cancer. Although its mechanism of photodynamic action is not well defined, A1PCTS is going to be under clinical trials of PDT. In this study, in vitro addition of A1PCTS to a suspension of rat epidermal microsomes followed by irradiation with red light (approximately 675 nm) resulted in significant destruction of cytochrome P-450 and associated monooxygenase activities. The photodestructive effect was dependent on both the dose of A1PCTS and the duration of light exposure. Studies using various quenchers of reactive oxygen species showed that only scavengers of singlet oxygen such as histidine, 2,5-dimethylfuran, beta-carotene and sodium azide afforded substantial protection against photodestruction. Our data indicate the direct involvement of singlet oxygen in the A1PCTS-mediated photodestructive process.     

Comprehensive report on the prevalence of root-knot nematodes in the Poonch division of Azad Jammu and Kashmir,Pakistan

The present study documents the root-knot nematodes (RKN) fauna of the Poonch division in Azad Jammu and Kashmir infecting vegetables. An overall prevalence of 40% of RKN was recorded. Of the four districts investigated, maximum prevalence was recorded in district Poonch with 59%, followed by Sudhnuti with 58%. The lowest prevalence of RKN was found in districts Bagh (29%) and Haveli (33%). Out of 15 vegetables investigated, RKN was found on five crops. The highest prevalence of 37.8% was recorded on okra, followed by 31.3% on cucumber and 17.5% on tomato. RKN was less prevalent on eggplant (8.3%) and beans (7.7%). Three RKN species, that is Meloidogyne incognita, Meloidogyne javanica and Meloidogyne arenaria, were found infecting the hosts. M. javanica was found to be the most prevalent followed by M. incognita and M. arenaria. This trend was found in all the districts. Overall prevalence of M. javanica as sole population was 9% and that of M. incognita was 2%. Meloidogyne arenaria was not found in any of the fields as sole population. The prevalence of M. incognita with M. javanica or M. arenaria as mixed populations was 8% and 5%, respectively, and that of M. javanica with M. arenaria was 4%. Similarly, all the three species prevailed as mixed populations in 12% of the fields in the division. The severity of RKN infections, measured as galling index, was found to be variable within each infected field (GI 2–9). Identification of RKN species was based on the morphology of perineal patterns and confirmed by molecular SCAR and CO1 makers based identification. In conclusion, RKN were distributed in the Poonch division and M. javanica was predominant. Cucumber, okra, tomato and eggplant were severely attacked by these nematodes warranting the adoption of stringent control strategies for their management.     

Structure-based prediction of protein–protein interactions between GhWlim5 Domain1 and GhACTIN-1 proteins: a practical evidence with improved fibre strength

Cotton fibre quality is a multigenic trait. Genetic modification of different genes to achieve high quality fibre is difficult without knowing the mechanism lying behind genes interaction. Based on background knowledge an attempt to explore the potential structural interactions between Gossypium hirsutum Wlim5 domain1 and Gossypium hirsutum ACTIN-1 proteins was done in current study. Sequence features of the LIM domain1 of GhWlim5 protein were identified through multiple sequence alignment analysis, and a phylogenetic tree was built to identify evolutionary relationships between sequences. Conservation indicated the evolutionary importance of side chain residues and the presence of several aliphatic and/or bulky residues, which stabilize the protein core and facilitate packing of zinc fingers. The structures of GhWlim5 domain1 and GhACTIN-1 proteins were modelled and validated through computational methods. Validation of GhACTIN-1 and GhWlim5 domain1 structures indicated good structural quality with 99.7% and 100% of the favoured number of residues in allowed regions and Z-score, within the ranges of − 9.87 and − 4.17, respectively. Docking analysis indicated various possible modes of interaction between these two proteins with favourable binding affinities. Based on our strong binding interaction results between GhWlim5 domain1 and GhACTIN-1 proteins, we further investigated the role of over-expression of GhWlim5 by transformation in cotton plants under fibre specific promoter and transgenic plants displayed significant increases in fibre strength.

     

Inhibition of cholesteryl ester transfer protein by anacetrapib does not impair the anti-inflammatory properties of high density lipoprotein

Cholesteryl ester transfer protein (CETP) is a target of therapeutic intervention for coronary heart disease. Anacetrapib, a potent inhibitor of CETP, has been shown to reduce LDL-cholesterol by 40% and increase HDL-cholesterol by 140% in patients, and is currently being evaluated in a phase III cardiovascular outcomes trial. HDL is known to possess anti-inflammatory properties, however with such large increases in HDL-cholesterol, it is unclear whether CETP inhibition perturbs HDL functionality such as anti-inflammatory effects on endothelial cells. The purpose of the present study was to determine whether CETP inhibition by anacetrapib affects the anti-inflammatory properties of HDL. HDL was isolated from either hamsters treated with vehicle or anacetrapib for 2 weeks, or from normal human subjects treated either placebo, 20 mg, or 150 mg anacetrapib daily for 2 weeks. Anacetrapib treatment increased plasma HDL cholesterol levels by 65% and between 48 and 82% in hamsters and humans, respectively. Pre-incubation of human aortic endothelial cells with HDL isolated from both control and anacetrapib treated hamsters suppressed TNFα induced expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and E-selectin. Similar results were obtained with human HDL samples pre and post treatment with placebo or anacetrapib. Further, HDL inhibited TNFα-induced MCP-1 secretion, monocyte adhesion and NF-κB activation in endothelial cells, and the inhibition was similar between control and anacetrapib treated groups. These studies demonstrate that anacetrapib treatment does not impair the ability of HDL to suppress an inflammatory response in endothelial cells.