The 4'-hydroxy group is responsible for the in vitro cytogenetic activity of resveratrol

We previously reported that 3,5,4'-trihydroxy-trans-stilbene (resveratrol), a polyphenolic phytoalexin found in grapes, induces a high frequency of sister chromatid exchanges (SCEs) in vitro. In this study, to investigate structure activity relationships, we synthesized six analogues of resveratrol differing in number and position of hydroxy groups, and we investigated their activity in chromosomal aberration (CA), micronucleus (MN) and sister chromatid exchange (SCE) tests in a Chinese hamster cell line (CHL). Two of the six analogues (3,4'-dihydroxy-trans-stilbene and 4-hydroxy-trans-stilbene) showed clear positive responses in a concentration-dependent manner in all three tests. Both were equal to or stronger than resveratrol in genotoxicity. The 4'-hydroxy (OH) analogue had the simplest chemical structure and was the most genotoxic. The other analogues did not have a 4'-hydroxy group. These results suggested that a 4'-hydroxy group is essential to the genotoxicity of stilbenes.     

Clastogenic effects of food additive citric acid in human peripheral lymphocytes

Clastogenic properties of the food additive citric acid, commonly used as an antioxidant, were analysed in human peripheral blood lymphocytes. Citric acid induced a significant increase of chromosomal aberrations (CAs) at all the concentrations and treatment periods tested. Citric acid significantly decreased mitotic index (MI) at 100 and 200 μg ml⁻¹ concentrations at 24 h, and in all concentrations at 48 h. However, it did not decrease the replication index (RI) significantly. Citric acid also significantly increased sister chromatid exchanges (SCEs) at 100 and 200 μg ml⁻¹ concentrations at 24 h, and in all concentrations at 48 h. This chemical significantly increased the micronuclei frequency (MN) compared to the negative control. It also decreased the cytokinesis-block proliferation index (CBPI), but this result was not statistically significant.     

The effects of 4-thujanol on chromosome aberrations,sister chromatid exchanges and micronucleus in human peripheral blood lymphocytes

4-Thujanol, a bicyclic monoterpene alcohol, is present in the essential oils of many medicinal and aromatic plants. It is commonly used as a fragrance and flavouring ingredient in a lot of different products. The potential genotoxic effects of 4-thujanol on human peripheral blood lymphocytes (PBLs) were investigated in vitro by the chromosome aberrations (CAs), sister chromatid exchanges (SCEs), and micronucleus (MN) tests. The cells were treated with 13, 26 and 52 μg/mL 4-thujanol in the presence and absence of a metabolic activator (S9 mix). 4-Thujanol induced CA (P < 0.001) and MN formation (P < 0.05) at all concentrations (13, 26 and 52 μg/mL) in the presence and absence of the S9 mix without a concentration-dependent manner. However, the treatment of peripheral lymphocytes with 4-thujanol did not produce a statistical difference in the frequency of SCEs when compared with control group. Furthermore, this monoterpene did not significantly decrease the mitotic index (MI), proliferation index (PI), and nuclear division index (NDI). In conclusion, 4-thujanol had a significant clastogenic effect at the tested concentrations (13, 26 and 52 μg/mL) for human PBLs. In addition, no cytotoxic and/or cytostatic effects were observed regardless of the concentrations used. This work presents the first report on genotoxic properties of 4-thujanol.     

Quantification of a potent mutagenic 4-amino-3,3′-dichloro-5,4′-dinitrobiphenyl (ADDB) and the related chemicals in water from the Waka River in Wakayama, Japan

4-Amino-3,3′-dichloro-5,4′-dinitrobiphenyl (ADDB) is a novel chemical exerting strong mutagenicity, especially in the absence of metabolic activation. In addition to mutagenicity, ADDB may also disrupt the endocrine system in vitro. ADDB may be discharged from chemical plants near the Waka River and could be unintentionally formed via post-emission modification of drainage water containing 3,3′-dichlorobenzidine (DCB), which is a precursor in the manufacture of polymers and dye intermediates in chemical plants. The main purpose of this study was to make a comprehensive survey of the behaviour and levels of ADDB and suspected starting material or intermediates of ADDB, i.e., DCB, 3,3′-dichloro-4,4′-dinitrobiphenyl (DDB), and 4-amino-3,3′-dichloro-4′-nitrobipheny (ADNB) in Waka River water samples. We also postulated the formation pathway of ADDB. Water samples were collected at five sampling sites from the Waka River four times between March 2003 and December 2004. Samples were passed through Supelpak2 columns, and adsorbed materials were then extracted with methanol. Extracts were used for quantification of ADDB and the related chemicals by HPLC on reverse-phase columns; mutagenicity was evaluated in the Salmonella assay using the O-acetyltransferase-overexpressing strain YG1024. High levels of ADDB, DCB, DDB, and ADNB (12.0, 20,400, 134.8, and 149.4 ng/L-equivalent) were detected in the samples collected at the site where wastewater was discharged from chemical plants into the river. These water samples also showed stronger mutagenicity in YG1024 both with and without S9 mix than the other water samples collected from upstream and downstream sites. The results suggest that ADDB is unintentionally formed from DCB via ADNB in the process of wastewater treatment of drainage water containing DCB from chemical plants.     

The role of 5′-methylthioadenosine phosphorylase in 5′-methylthioadenosine-mediated inhibition of lymphocyte transformation

To determine if increased 5′-methylthioadenosine phosphorylase activity in activated lymphocytes may be responsible for the decreased inhibitory effect noted when 5′-methylthioadenosine is added after stimulation, the activity of this enzyme was monitored during lymphocyte transformation. A direct correlation existed between the transformation process and 5′-methylthioadenosine phosphorylase activity; the longer the stimulation process progressed, the greater the enzyme activity. The 7-deaza analog of 5′-methylthioadenosine, 5′-methylthiotubercidin, was utilized to explore further the role that the phosphorylase may play in the reversal process. 5′-Methylthioadenosine acted as a potent inhibitor, but not a substrate, of the 5′-methylthioadenosine phosphorylase, and was an even more potent inhibitor of lymphocyte transformation than 5′-methylthioadenosine. However, in direct contrast to the 5′-methylthioadenosine effect, inhibition by 5′-methylthiotubercidin could not be completely reversed. These data suggest the 5′-methylthioadenosine phosphorylase plays an important role in reversing 5′-methylthioadenosine-mediated inhibition and that the potent, nonreversible inhibitory effects of 5′-methylthiotubercidin are due to its resistance to 5′-methylthioadenosine phosphorylase degradation.     

High-performance liquid chromatographic assay for the determination of 2′-deoxy-3′-thiacytidine (lamivudine) in human plasma

A method for the quantification of 2′-deoxy-3′-thiacytidine (lamivudine, 3-TC), which incorporated the use of 3-isobutyl-methylxanthine as internal standard (I.S.) was developed and validated in human plasma, using HPLC with UV absorbance detection. Using solid-phase extraction, 3-TC and I.S. were selectively extracted from human plasma. Subsequently, chromatographic separation was performed using a YMC phenyl column with ion-pair chromatography and detection at 270 nm. The method was validated over a concentration range of 10 to 5000 ng/ml using 0.5 ml of human plasma. The extraction recovery for both 3-TC and I.S. was greater than 95%. The determination of inter- and intra-day precision (RSD) was less than 10% at all concentration levels, while the inter- and intra-day accuracy (% difference) was less than 6%.     

Rapid quantitation of (−)-2′-deoxy-3′-thiacytidine in human serum by high-performance liquid chromatography with ultraviolet detection

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed and validated for the measurement of (−)-2′-deoxy-3′-thiacytidine (3TC) in human serum. The method included precipitation of serum proteins by trichloroacetic acid (20%, w/v) treatment followed by centrifugation. The resulting supernatant was directly injected and 3TC was isocratically chromatographed on a reversed-phase C₁₈ column using a mixture of phosphate buffer and methanol (88.3:11.7, v/v) and monitored at 280 nm. The limit of quantitation was 20 ng/ml using 100 μl of serum. The standard curve was linear within the range of 20–10 000 ng/ml. Replicate analysis of three quality control samples (40–1500 ng/ml) led to satisfactory intra- and itner-assay precision (coefficient of variation from 3.0 to 12.9%) and accuracy (deviation from −6.3 to 9.7%). Moreover, sample treatment processes including human immunodeficiency virus (HIV) heat-inactivation, exposure at room temperature and freezing-thawing cycles did not influence the stability of the analyte. This assay was successfully applied to the determination of 3TC serum levels in HIV-infected patients. In addition, preliminary results indicated that this procedure may also be extended to the measurement of 3TC in human plasma and urine.     

Micronuclei in peripheral lymphocytes and exfoliated urothelial cells of workers exposed to 4,4′-methylenebis-(2-chloroaniline) (MOCA)

4,4′-Methylenebis-(2-chloroaniline) (MOCA) is used in the manufacture of polyurethane. The IARC classifies MOCA as a probable human carcinogen. Suggested changes to guidelines for health surveillance of MOCA-exposed workers in Australia include a reduction in acceptable levels of urinary MOCA to below 15 μmol/mol creatinine. Twelve male workers aged 24 and 42 years were recruited into this study from four work locations where MOCA is used. Exfoliated urothelial cells from prework urine samples on a midweek work day were assessed for micronucleus (MN) frequencies. Postwork urine samples were analysed for total MOCA. Blood samples collected on the same day were cultured for 96 h and cytochalasin-B-blocked cells were scored for MN. Eighteen male control subjects (23–59 years) provided corresponding urine and blood samples. Median urinary MOCA concentrations were 6.5 μmol/mol creatinine (range 0.4–48.6 μmol/mol creatinine) in postwork samples of MOCA-exposed workers. MOCA was not detected in urine of control workers. Mean MN frequencies were higher in urothelial cells and lymphocytes of MOCA workers (14.27±0.56 and 13.25±0.48 MN/1000 cells) than in controls (6.90±0.18 and 9.24±0.29 MN/1000 cells). The mean number of micronucleate cells was also higher in both tissues of exposed workers (9.69±0.32 and 8.54±0.14 MN cells/1000 cells) than in controls (5.18±0.11 and 5.93±0.13 MN cells/1000). There was no correlation between postwork urinary MOCA concentrations and MN frequencies in either tissue. This study suggests that exposures to MOCA in South Australia are similar to those of a decade ago and are at levels similar to those currently acceptable in Australia. These are associated with genotoxic effects in urothelial cells and peripheral blood lymphocytes. It may be prudent to reduce MOCA exposures in line with proposed guidance values.     

Nuclear Location of a Diadenosine 5′,5′”-P1,P4Tetraphosphate (Ap4A) Hydrolase in Tomato Cells Grown in Suspension Cultures1

Diadenosine 5′,5′”-P¹,P⁴-tetraphosphate (Ap₄A) cleaving enzymes are assumed to regulate intracellular levels of Ap₄A, a compound known to affect cell proliferation and stress responses. From plants an Ap₄A hydrolase was recently purified using tomato cells grown in suspension. It was partially sequenced and a peptide antibody was prepared (Feussner et al., 1996). Using this polyclonal monospecific antibody, an abundant nuclear location of Ap₄A hydrolase in 4-day-old cells of atomato cell suspension culture is demonstrated here by means of immunocytochemical techniques using FITC (fluorescein-5-isothiocyanate) labeled secondary antibodies. The microscopic analysis of the occurrence of Ap₄A hydrolase performed for different stages of the cell cycle visualized by parallel DAPI (4,6-diamidino-2-phenylindole) staining revealed that the protein accumulates within nuclei of cells in the interphase, but is absent in the nucleus as well as cytoplasm during all stages of mitosis. This first intracellular localization of an Ap₄A degrading enzyme within the nucleus and its pattern of appearance during the cell cycle is discussed in relation to the suggested role of Ap₄A in triggering DNA synthesis and cell proliferation.     

Increased 3′‐Phosphoadenosine‐5′‐phosphosulfate Levels in Engineered Escherichia coli Cell Lysate Facilitate the In Vitro Synthesis of Chondroitin Sulfate A

Chondroitin sulfates (CSs) are linear glycosaminoglycans that have important applications in the medical and food industries. Engineering bacteria for the microbial production of CS will facilitate a one‐step, scalable production with good control over sulfation levels and positions in contrast to extraction from animal sources. To achieve this goal, Escherichia coli (E. coli) is engineered in this study using traditional metabolic engineering approaches to accumulate 3′‐phosphoadenosine‐5′‐phosphosulfate (PAPS), the universal sulfate donor. PAPS is one of the least‐explored components required for the biosynthesis of CS. The resulting engineered E. coli strain shows an ≈1000‐fold increase in intracellular PAPS concentrations. This study also reports, for the first time, in vitro biotransformation of CS using PAPS, chondroitin, and chondroitin‐4‐sulfotransferase (C4ST), all synthesized from different engineered E. coli strains. A 10.4‐fold increase is observed in the amount of CS produced by biotransformation by employing PAPS from the engineered PAPS‐accumulating strain. The data from the biotransformation experiments also help evaluate the reaction components that need improved production to achieve a one‐step microbial synthesis of CS. This will provide a new platform to produce CS.     

Cyclic adenosine 5′‐diphosphoribose (cADPR) cyclic guanosine 3′,5′‐monophosphate positively function in Ca2+ elevation in methyl jasmonate‐induced stomatal closure,cADPR is required for methyl jasmonate‐induced ROS accumulation NO production in guard cells

Methyl jasmonate (MeJA) signalling shares several signal components with abscisic acid (ABA) signalling in guard cells. Cyclic adenosine 5′‐diphosphoribose (cADPR) and cyclic guanosine 3′,5′‐monophosphate (cGMP) are second messengers in ABA‐induced stomatal closure. In order to clarify involvement of cADPR and cGMP in MeJA‐induced stomatal closure in Arabidopsis thaliana (Col‐0), we investigated effects of an inhibitor of cADPR synthesis, nicotinamide (NA), and an inhibitor of cGMP synthesis, LY83583 (LY, 6‐anilino‐5,8‐quinolinedione), on MeJA‐induced stomatal closure. Treatment with NA and LY inhibited MeJA‐induced stomatal closure. NA inhibited MeJA‐induced reactive oxygen species (ROS) accumulation and nitric oxide (NO) production in guard cells. NA and LY suppressed transient elevations elicited by MeJA in cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) in guard cells. These results suggest that cADPR and cGMP positively function in [Ca²⁺]_cyt elevation in MeJA‐induced stomatal closure, are signalling components shared with ABA‐induced stomatal closure in Arabidopsis, and that cADPR is required for MeJA‐induced ROS accumulation and NO production in Arabidopsis guard cells.     

A method for rapid and sensitive negative staining of proteins in SDS‐PAGE using 2′,7′‐dichlorofluorescein

We report here a rapid and sensitive technique for negative visualization of protein in 1D and 2D SDS‐PAGE by using 2′, 7′‐dichlorofluorescein (DCF), which appeared as transparent and colorless bands in an opaque gel matrix background. For DCF stain, down to 0.1–0.2 ng protein could be easily visualized within 7 min by only two steps, and the staining is fourfold more sensitive than that of Eosin Y (EY) negative stain and glutaraldehyde (GA) silver stain, and eightfold more sensitive than that of the commonly used imidazole‐zinc (IZ) negative stain. Furthermore, DCF stain provided good reproducibility, linearity, and MS compatibility compared with those of IZ stain. In addition, the potential staining mechanism was investigated by colorimetric experiment and molecular docking, and the results demonstrated that the interaction between DCF and protein occurs mainly via van der waals force, electrostatic interaction, and hydrogen bonding.     

1,3-dihydroxy-5-(tridec-4′,7′-dienyl)benzene: a new cytotoxic compound from Lithraea molleoides

A dichloromethane extract from the leaves of Lithraea molleoides (Anacardiaceae), an argentine medicinal plant, showed cytotoxicity on human hepatocellular carcinoma cell line. Bioassay guided fractionation of this extract led to the isolation of a new active 5-alkyl resorcinol: 1,3-dihydroxy-5-(tridec-4',7'-dienyl)benzene. Chemical structure was established based on spectroscopic data (UV, IR, MS, 1H-NMR, 13C-NMR, COSY). This compound presented cytotoxic activity on 3 human tumoral cell lines: hepatocellular carcinoma cell line-Hep G2 (IC50 +/- SD of 68 +/- 2 microM), mucoepidermoid pulmonary carcinoma cell line-H292 (IC50 +/- SD of 63 +/- 5 microM) and mammary gland adenocarcinoma cell line -MCF7 (IC50 +/- SD of 147 +/- 5).     

Differential modulation of hepatic cytochrome P-450 enzymes in rat and syrian hamster by 4′-trifluoromethyl-2,3,4,5-tetrachlorobiphenyl

The effects of a single injection (40 mg/kg) of 4′-trifluoromethyl-2,3,4,5-tetrachlorobiphenyl (CF3) on hepatic cytochrome P-450 monooxygenases were assessed in rat and syrian hamster. The CF3 treatment significantly increased the total amount of cytochrome P-450 in both species. In rats, CF3 treatment caused marked increases in ethoxyresorufin O-deethylase (EROD), arylhydrocarbon hydroxylase (AHH), and testosterone 7α-hydroxylase activities but significantly reduced the activities of benzphetamine N-demethylase (BzND), erythromycin N-demethylase (ErND), testosterone 6β, 16α, and 16β-hydroxylases, and formation of androstenedione. Administration of CF3 to hamsters strongly induced the activities of EROD, AHH, BzND, testosterone 15α, and 16α-hydroxylases, and androstenedione production, whereas ErND, testosterone 6β, and 7α-hydroxylases were decreased. Administration of CF3 to rats induced the CYP1A family proteins and CYP2A1, while CF3 reduced the level of CYP2B1, and, to a lesser extent, of CYP6β2. In hamsters, CF3 treatment significantly induced the CYP1A2, CYP2A1, CYP2A8, and CYP2B1 isozymes, whereas the CYP6β2 level was decreased. The ability of hepatic microsomes to activate aflatoxin B1 and benzo(a)pyrene was elevated by CF3 treatment in hamsters, while activation of aflatoxin B1 was decreased in microsomes from CF3-treated rats. These results showed differences in the CF3-induced pattern of rat and hamster cytochrome P-450 monooxygenases.     

Electrochemiluminescence of tris(2,2′‐bipyridyl)ruthenium and its applications in bioanalysis: a review

Electrochemiluminescence (ECL) of tris(2,2’‐bipyridyl)ruthenium(II) [Ru(bpy)₃²⁺] is an active research area and includes the synthesis of ECL‐active materials, mechanistic studies and broad applications. Extensive research has been focused on this area, due to its scientific and practical importance. In this mini‐review we focus on the bio‐related applications of ECL. After a brief introduction to Ru(bpy)₃²⁺ ECL and its mechanisms, its application in constructing an effective bioassay is discussed in detail. Three types of ECL assay are covered: DNA, immunoassay and functional nucleic acid sensors. Finally, future directions for these assays are discussed. Copyright © 2011 John Wiley & Sons, Ltd.     

Effects of P450 inhibition and induction on the olfactory toxicity of β, β′-iminodipropionitrile (IDPN) in the rat

In addition to the neurotoxic effects of β, β′-iminodipropionitrile (IDPN) which have been previously reported by other investigators, the olfactory toxicity of this compound has recently been uncovered in this laboratory. Due to the apparently conflicting observations that the IDPN-induced lesion in the olfactory mucosa is very focal in nature (suggesting site-specific activation) and the observation by other investigators that the behavioral effects of IDPN appear to be due to the parent compound, we initiated studies into the possible role of the cytochrome P450 enzymes in the olfactory toxicity of IDPN. Immunohistochemical studies with antibodies raised against several different P450 isoforms revealed good correlation between IDPN-induced olfactory mucosal degeneration and the localization of a protein immunoreacting with an antibody to P450 2E1. Enzymatic studies revealed that there is approximately fivefold more ρ-nitrophenol hydroxylation activity in the olfactory mucosa than in the liver on a per milligram microsomal protein basis. Administration of 1% acetone in the drinking water increased the levels of olfactory mucosal 2E1, and the increase in enzyme levels corresponded to increased olfactory toxicity of IDPN; inhibition of P450 activities with either metyrapone or carbon tetrachloride eliminated or significantly decreased the olfactory toxicity of IDPN, respectively. These studies suggest a role for cytochrome P450, specifically the 2E1 isoform, in the activation of IDPN within the nasal mucosa.     

Modified high-performance liquid chromatography assay for the measurement of 2′-deoxyuridine in human plasma and its application to pharmacodynamic studies of antimetabolite drugs

A new method is presented for the HPLC determination of plasma 2′-deoxyuridine (dUrd). Briefly, 1 ml of human plasma is deproteinised with perchloric acid followed by purification by solid-phase extraction using a non-polar high-capacity polymeric sorbent. The dUrd is separated on a C₁₈ reversed-phase column using a mobile-phase of 0.05% v/v trifluoroacetic acid in water, with a retention time of 8.5 min at a flow-rate of 1.25 ml min⁻¹. Quantitation is by UV detection at 261 nm using a photodiode array detector. The limit of quantitation is 6 nM with a linear response over the measured range 6–400 nM. Both intra- and inter-day RSD and bias are typically less than 13%. Chromatograms and pharmacodynamic data from a Phase 1 Clinical Trial of a new antifolate drug, ZD9331 are included to illustrate the utility of the method. They show the increase in circulating dUrd as a result of drug inhibition of the target enzyme thymidylate synthase. The method has the significant advantages of ease and simplicity over earlier methods and may be applied to the analysis of other nucleoside species.     

Flow‐injection analysis for meloxicam based on tris(2,2′‐bipyridine) ruthenium(II)–Ce(IV) chemiluminescent system

It was found that meloxicam could enhance the chemiluminescence (CL) of the tris(2,2'‐bipyridine) ruthenium(II)–Ce(IV) system in the medium of sulfate acid. Based on this phenomenon a new flow‐injection system with chemiluminescent detection has been proposed for determination of meloxicam. Under optimum conditions, meloxicam had a good linear relationship with the CL intensity in the concentration range of 6.0  10^?4 to 1.0 µg/mL and the detection limit was 3.7 × 10^?4 µg/mL. The proposed method was applied to detect meloxicam in tablets and a satisfactory recovery was obtained. The possible mechanism for this CL system is also discussed in this paper. Copyright © 2009 John Wiley & Sons, Ltd.