DNA alkylation lesions and their repair in human cells: modification of the comet assay with 3-methyladenine DNA glycosylase (AlkD)

3-Methyladenine DNA glycosylase (AlkD) belongs to a new family of DNA glycosylases; it initiates repair of cytotoxic and promutagenic alkylated bases (its main substrates being 3-methyladenine and 7-methylguanine). The modification of the comet assay (single cell gel electrophoresis) using AlkD enzyme thus allows assessment of specific DNA alkylation lesions. The resulting baseless sugars are alkali-labile, and under the conditions of the alkaline comet assay they appear as DNA strand breaks. The alkylating agent methyl methanesulfonate (MMS) was used to induce alkylation lesions and to optimize conditions for the modified comet assay method with AlkD on human lymphoblastoid (TK6) cells. We also studied cellular and in vitro DNA repair of alkylated bases in DNA in TK6 cells after treatment with MMS. Results from cellular repair indicate that 50% of DNA alkylation is repaired in the first 60 min. The in vitro repair assay shows that while AlkD recognises most alkylation lesions after 60 min, a cell extract from TK6 cells recognises most of the MMS-induced DNA adducts already in the first 15 min of incubation, with maximum detection of lesions after 60 min’ incubation. Additionally, we tested the in vitro repair capacity of human lymphocyte extracts from 5 individuals and found them to be able to incise DNA alkylations in the same range as AlkD. The modification of the comet assay with AlkD can be useful for in vitro and in vivo genotoxicity studies to detect alkylation damage and repair and also for human biomonitoring and molecular epidemiology studies.     

Study of gene-specific DNA repair in the comet assay with padlock probes and rolling circle amplification

We used padlock probes to study the rate of gene specific repair of three genes, OGG1 (8-oxoguanine-DNA glycosylase-1), XPD (xeroderma pigmentosum group D), and HPRT (hypoxanthine-guanine phosphoribosyltransferase) in human lymphocytes, in relation to the repair rate of Alu repeats and total genomic DNA. Padlock probes offer highly specific detection of short target sequences by combining detection by ligation and signal amplification. In this approach only genes in sequences containing strand breaks, which become single-stranded in the tail, are available for hybridisation. Thus the total number of signals from the padlock probes per comet gives a direct measure of the amount of damage (strand-breaks) present and allows the repair process to be monitored. This method could provide insights on the organisation of genomic DNA in the comet tail. Alu repeat containing DNA was repaired rapidly in comparison with total genomic DNA, and the studied genes were generally repaired more rapidly than the Alu repeats.     

Recent advances into the pathogenesis of recurrent urinary tract infections: the bladder as a reservoir for uropathogenic Escherichia coli

Recurrent cystitis is a common clinical entity that is becoming increasingly challenging to manage. The current thought regarding the pathogenesis of these infections is evolving as new data emerges to suggest that some recurrences may originate from previously unrecognized reservoirs of uropathogenic Escherichia coli. This article will summarize recent conceptual changes with respect to the aetiology of recurrent urinary tract infections, particularly as it relates to chronic bacterial persistence in the bladder.     

Genotyping DNA chip for the simultaneous assessment of antibiotic resistance and pathogenic potential of extraintestinal pathogenic Escherichia coli

Urinary tract infections (UTIs) are among the most frequently occurring infections and are mostly caused by extraintestinal pathogenic Escherichia coli. DNA microarrays are potent molecular diagnostic tools for rapid diagnosis of bacterial infections with high relevance for UTIs. In this study, we present the integration and application of two DNA chip modules for the simultaneous detection of single nucleotide polymorphisms in gyrA (quinolone resistance) and fimH (increased adhesion to urinary tract epithelium). The performance of the combined diagnostic chip was assessed by genotyping 140 E. coli strains. Resistance-causing mutations could only be identified in UTI isolates. A complete genotyping assay could be performed in <4 h after DNA extraction. Together with the excellent genotyping results, this constitutes a competitive alternative as a standard tool for routine clinical diagnostics.     

The association of minocycline and the probiotic Escherichia coli Nissle 1917 results in an additive beneficial effect in a DSS model of reactivated colitis in mice

Antibiotics have been empirically used for human inflammatory bowel disease, being limited to short periods. Probiotics are able to attenuate intestinal inflammation due to its immunomodulatory properties, being considered as safe when chronically administered. The aim was to test the association of minocycline, a tetracycline with immunomodulatory properties, and the probiotic Escherichia coli Nissle 1917 (EcN) in a mouse model of reactivated colitis. For this purpose, female C57BL/6J mice were assigned to different groups: non-colitic and dextran sodium sulfate (DSS)-control groups (without treatment), minocycline (50 mg/kg/day; p.o.), EcN (5 × 10⁸ CFU/day; p.o.), and minocycline plus EcN treated groups. Colitis was induced by adding DSS in the drinking water (3%) for 5 days; 2 weeks later, colitis was reactivated by subsequent exposure to DSS. The inflammatory status was evaluated daily by a disease activity index (DAI); colonic damage was assessed histologically and biochemically by evaluating mRNA relative expression of different mediators by qPCR. Finally, a microbiological analysis of the colonic contents was performed. Minocycline and EcN exerted intestinal anti-inflammatory effect and attenuated the reactivation of the colitis, as shown by the reduced DAI values, being these effects greater when combining both treatments. This was evidenced histologically and biochemically, by reduced expression of TNFα, IL-1β, IL-2, MIP-2, MCP-1, ICAM-1, iNOS and MMP-9, together with increased MUC-3 and ZO-1 expression. Finally, the altered microbiota composition of colitic mice was partially restored after the different treatments. In conclusion, EcN supplementation to minocycline treatment improves the recovery of the intestinal damage and prevents the reactivation of experimental colitis.     

Evaluation of the genotoxic potential of single-wall carbon nanotubes by using a battery of in vitro and in vivo genotoxicity assays

The genotoxic potential of a high purity sample of single-wall carbon nanotubes (SWCNTs) was evaluated using a battery of in vitro and in vivo genotoxicity assays. These comprised a bacterial reverse mutation test (Ames test), an in vitro chromosomal aberration test, and an in vivo mouse bone marrow micronucleus test. The SWCNTs exerted no genotoxicity in Salmonella typhimurium TA97, TA98, TA100, and TA1535, or in Escherichia coli WP2 uvrA/pKM101, whether in the absence or presence of metabolic activation and at concentrations of 12.5–500 μg/plate. In the chromosomal aberration test, at 300–1000 μg/mL, the SWCNTs did not increase the number of structural or numerical chromosomal aberrations, whether the test was conducted with or without metabolic activation. In the in vivo bone marrow micronucleus test, doses of 60 mg/kg and 200 mg/kg SWCNTs did not affect the proportions of immature and total erythrocytes, nor did it increase the number of micronuclei in the immature erythrocytes of mice. The results of these studies show that the high purity and well-dispersed sample of SWCNTs are not genotoxic under the conditions of the in vitro bacterial reverse mutation assay, chromosomal aberration assay, or in vivo bone marrow micronucleus test, and thus appear not to pose a genotoxic risk to human health in vivo.     

Assessment of the genotoxic and carcinogenic risks of p-nitrophenol when it is present as an impurity in a drug product

According to the 2008 US FDA (draft) and 2006 EMEA guidance documents for genotoxic impurities, an impurity that is positive in an in vitro genotoxicity study, in the absence of in vivo genotoxicity or carcinogenicity data, should be treated as genotoxic and typically controlled to 1.5 μg/day for chronic use. For p-nitrophenol (PNP), existing study results (i.e., positive in vitro clastogenicity in mammalian cells, no information on its in vivo genotoxicity, and negative with respect to carcinogenicity in a dermal mouse study with no confirmation of systemic exposure) indicated that it should be considered genotoxic and exposure as a drug impurity limited. Therefore, to more completely characterize the genotoxic potential of PNP (consistent with the guidance documents), in vivo mouse micronucleus and dermal pharmacokinetic bridging studies were conducted. In the micronucleus study, PNP was negative, demonstrating that the reported in vitro clastogenicity is not present in vivo. In the pharmacokinetic study, PNP was well absorbed dermally, validating the negative dermal carcinogenicity assessment. These results indicate that PNP should be considered a non-genotoxic impurity and, as a drug impurity, a threshold limit of 4 mg/day would be set (per ICH Q3C). This threshold limit is higher than the EPA reference dose (listed in the 2006 Edition of the Drinking Water Standards and Health Advisories), so if present at such levels, the specification limits for PNP should be determined on a case-by-case basis, based on risk-benefit.     

A Simple and Rapid Procedure for the Detection of Genes Encoding Shiga Toxins and Other Specific DNA Sequences

A novel procedure for the detection of specific DNA sequences has been developed. This procedure is based on the already known method employing PCR with appropriate primers and a sequence-specific DNA probe labeled with the fluorescent agent 6-carboxylfluorescein (FAM) at the 5′ end and the fluorescence quencher BHQ-1 (black hole quencher) at the 3′ end. However, instead of the detection of the fluorescence signal with the use of real-time PCR cyclers, fluorescence/luminescence spectrometers or fluorescence polarization readers, as in all previously-reported procedures, we propose visual observation of the fluorescence under UV light directly in the reaction tube. An example for the specific detection of the Shiga toxin-producing Escherichia coli (STEC) strains, by detecting Shiga toxin genes, is demonstrated. This method appears to be specific, simple, rapid and cost effective. It may be suitable for use in research laboratories, as well as in diagnostic units of medical institutions, even those equipped only with a thermocycler and a UV transilluminator, particularly if rapid identification of a pathogen is required.     

Identification of <Emphasis Type="Italic">Curcuma</Emphasis> plants and curcumin content level by DNA polymorphisms in the <Emphasis Type="Italic">trn</Emphasis>S-<Emphasis Type="Italic">trn</Emphasis>fM intergenic spacer in chloroplast DNA

With the goal of developing an accurate plant identification method, molecular analysis based on polymorphisms of the nucleotide sequence of chloroplast DNA (cpDNA) was performed in order to distinguish four Curcuma species: C. longa, C. aromatica, C. zedoaria, and C. xanthorrhiza. Nineteen regions of cpDNA were amplified successfully via polymerase chain reaction (PCR) using total DNA of all Curcuma plants. Using the intergenic spacer between trnS and trnfM (trnSfM), all four Curcuma plant species were correctly identified. In addition, the number of AT repeats in the trnSfM region was predictive of the curcumin content in the rhizome of C. longa.     

Development of a rapid and sensitive LC-MS/MS assay for the determination of sorafenib in human plasma

A rapid and sensitive liquid chromatography/tandem mass spectrometric (LC/MS/MS) assay was developed for the quantitative determination of sorafenib in human plasma. Sample pretreatment involved simple protein precipitation by the addition of 0.5 mL acetonitrile, containing internal standard ([2H3, 15N] sorafenib), to 50 microL of plasma sample volume. Separation was achieved on a Waters SymmetryShield RP8 (2.1 mm x 50 mm, 3.5 microm) column at room temperature using an isocratic elution method with acetonitrile/0.1% formic acid in water: 65/35 (v/v) at a flow rate of 0.25 mL/min. Detection was performed using electrospray ionization in positive ion multiple reaction monitoring (MRM) mode by monitoring the ion transitions from m/z 464.9 --> 252.0 (sorafenib) and m/z 469.0 --> 259.0 (internal standard). Calibration curves were linear in the concentration range of 5-2000 ng/mL. The accuracy and precision values, calculated from three different sets of quality control samples analyzed in quintuplicate on six different days, ranged from 92.86% to 99.88% and from 1.19% to 4.53%, respectively.     

Lack of in vivo mutagenicity and oxidative DNA damage by flumequine in the livers of <Emphasis Type="Italic">gpt</Emphasis> delta mice

Flumequine (FLU), an anti-bacterial quinolone agent, has been recognized as a non-genotoxic carcinogen for the mouse liver, but recent reports have suggested that some genotoxic mechanism involving oxidative DNA damage may be responsible for its hepatocarcinogenesis. In the present study, we investigated this possibility in the mouse liver using male and female B6C3F1 gpt delta mice fed diet containing 0.4% FLU, a carcinogenic dose, for 13 weeks. Measurements of 8-hydroxydeoxyguanosine levels in liver DNA, and gpt point and deletion mutations revealed no significant increases in any of these parameters in either sex. Histopathologically, centrilobular swelling of hepatocytes with vacuolation was apparent, however, together with significant increase in bromodeoxyuridine-labeling indices in the treated males and females. These results suggest that genotoxicity, including oxidative DNA damage, is not involved in mouse hepatocarcinogenesis by FLU, which might rather solely exert tumor-promoting effects in the liver.     

In vitro and in vivo assessment of the genotoxic activity of aloesin

Aloesin is a chromone that is a component of Aloe spp. It may have potential as a functional food ingredient as it has been shown to likely have beneficial effects in persons in a pre-diabetic state or who have metabolic syndrome. In this study the safety of aloesin has been evaluated using a series of in vitro and in vivo genotoxicity assays including, bacterial mutation, mammalian cell cytogenetic, and mouse micronucleus tests. Aloesin did not induce reverse mutations in Salmonella typhimurium and Escherichia coli at any of the tested dose levels up to 10,000 μg/plate. Similarly, aloesin did not increase the incidence of chromosome aberrations when incubated with Chinese hamster lung cells at any of the tested concentrations up to 10,000 μg/mL. In vivo, there was no effect of aloesin on the incidence of micronucleated erythrocytes following oral administration on two consecutive days at doses up to 5000 mg/kg body weight. There was no evidence of toxicity to bone marrow. The results of these studies demonstrate that aloesin is without genotoxic potential.     

Development and full validation of a sensitive quantitative assay for the determination of clemastine in human plasma by liquid chromatography-tandem mass spectrometry

A sensitive high-performance liquid chromatography-tandem mass spectrometry (LC/MS/MS) method was developed and validated for the determination of clemastine in human plasma. After having been extracted from plasma samples by ethyl acetate, clemastine and internal standard, diphenhydramine, were separated on a C(18) column. Detection was performed on Thermo Finnigan TSQ Quantum triple quadrupole mass spectrometer by selected reaction monitoring (SRM) mode via electrospray ionization (ESI) source. The method was linear in the concentration range of 5.0-1000.0 pg/ml for clemastine. The intra- and inter-day precisions were within 13.4% and the deviations were between -1.1% and 5.6%. The fully validated LC/ESI-MS/MS method has been successfully applied to the preliminary pharmacokinetic study in healthy male Chinese volunteers.     

Role of retinoic acid in the modulation of benzo(a)pyrene-DNA adducts in human hepatoma cells: implications for cancer prevention

Carcinogen-DNA adducts could lead to mutations in critical genes, eventually resulting in cancer. Many studies have shown that retinoic acid (RA) plays an important role in inducing cell apoptosis. Here we have tested the hypothesis that levels of carcinogen-DNA adducts can be diminished by DNA repair and/or by eliminating damaged cells through apoptosis. Our results showed that the levels of total DNA adducts in HepG2 cells treated with benzo(a)pyrene (BP, 2 μM) + RA (1 μM) were significantly reduced compared to those treated with BP only (P = 0.038). In order to understand the mechanism of attenuation of DNA adducts, further experiments were performed. Cells were treated with BP (4 μM) for 24 h to initiate DNA adduct formation, following which the medium containing BP was removed, and fresh medium containing 1 μM RA was added. The cells were harvested 24 h after RA treatment. Interestingly, the levels of total DNA adducts were lower in the BP/RA group (390 ± 34) than those in the BP/DMSO group (544 ± 33), P = 0.032. Analysis of cell apoptosis showed an increase in BP + RA group, compared to BP or RA only groups. Our results also indicated that attenuation of BP-DNA adducts by RA was not primarily due to its effects on CYP1A1 expression. In conclusion, our results suggest a mechanistic link between cellular apoptosis and DNA adduct formation, phenomena that play important roles in BP-mediated carcinogenesis. Furthermore, these results help understand the mechanisms of carcinogenesis, especially in relation to the chemopreventive properties of nutritional apoptosis inducers.     

Spectrophotometric determination of pyrrole-like substances in urine of rat and man: An assay for the evaluation of 2,5-hexanedione formed fromn-hexane

Male Wistar rats exposed to atmosphericn-hexane excreted in their urine substances which gave rise to absorption spectra like those of pyrroles after the reaction with Ehrlich's reagent. A simple spectrophometric assay was developed to determine these pyrrole-like substances in urine. Their excretion kinetics were evaluated by exposing rats for 8 h to atmosphericn-hexane concentrations between 50 and 3000 ppm. The dose-response curve revealed saturation kinetics according to Michaelis-Menten, V_max being 1.12 [E ⁵²⁶ml urine/8 hn-hexane exposure] and Km, the atmosphericn-hexane concentration at V_max/2, being 250 ppm. The excretion of pyrrolelike substances closely correlated with that of 2,5-hexanedione measured by Fedtke and Bolt (1987). Pyrrole-like substances were also found in the urine of a male volunteer. When exposing the person for 3 h to atmosphericn-hexane at a concentration of 146 ppm (equivalent to 55 ppm/8 h) the excreted amount was twice the background value. Due to the sensitivity of this assay it is possible to determine pyrrole-like substances in urine according to the present German MAK or US TLV conditions forn-hexane (50 ppm/8 h).     

Oxidative DNA damage and in vivo mutagenicity caused by reactive oxygen species generated in the livers of p53‐proficient or ‐deficient gpt delta mice treated with non‐genotoxic hepatocarcinogens

Oxidative stress is thought to participate in chemical carcinogenesis and may trigger gene mutations. To accurately assess the carcinogenesis risk posed to humans by chemical exposure, it is important to understand the pathways by which reactive oxygen species (ROS) are generated and the effects of the resulting oxidative stress. In the present study, p53‐proficient and ‐deficient gpt delta mice were given pentachlorophenol (PCP), phenobarbital (PhB) or piperonyl butoxide (PBO), which are classified as non‐genotoxic hepatocarcinogens in rodents, at the respective carcinogenic doses for 13 weeks. Exposure to PCP or PBO, but not PhB, invoked significant increases in liver DNA 8‐hydroxydeoxyguanosine (8‐OHdG) levels. Treatment with PCP significantly increased mRNA levels of the gene encoding NAD(P):quinone oxidoreductase 1 (NQO1) in the liver, suggesting that redox cycling of the PCP metabolite tetrachlorohydroquinone gave rise to ROS. Exposure to PhB or PBO significantly elevated CYP 2B10 mRNA levels while NQO1 levels were also significantly increased in PBO‐treated mice. Therefore, in addition to involvement of the CYP catalytic pathway in the ROS‐generated system of PBO, catechol derivatives produced from the opening of the PBO functional group methylenedioxy ring probably resulted in ROS generation. However, PCP, PBO and PhB failed to increase gpt and red/gam gene mutations in the liver independently of p53. Overall, the action of oxidative stress by ROS derived from the metabolism of these carcinogens might be limited to cancer‐promoting activity, which supports the previous classification of these carcinogens as non‐genotoxic. Copyright © 2012 John Wiley & Sons, Ltd.     

Development of a simple, rapid and reproducible HPLC assay for the simultaneous determination of hypericins and stabilized hyperforin in commercial St. John's wort preparations

A reversed-phase HPLC method was developed and validated for the simultaneous determination of hypericins and stabilized hyperforin in St. John's Wort extract. The sample solution was prepared by extraction of the finely powdered extract with methanol–water (80:20, v/v) containing 5% HP-β-cyclodextrin, and adjusted to pH 2.5 with orthophosphoric acid. Diluted extract solutions, maintained at 0 °C, were injected into a C₁₈ column. The samples were eluted isocratically using a mobile phase consisting of acetonitrile and 0.3% v/v phosphoric acid (90:10, v/v) at a 1.5 ml/min flow rate with simultaneous fluorescence (315/590 nm, excitation/emission) and UV (273 nm) detection. Quantification of the marker compounds (hypericin, pseudohypericin, hyperforin) was achieved by use of standard curves generated by plotting peak heights versus concentrations. Validation studies demonstrated that this HPLC method is simple, rapid, reliable, and reproducible. The standard curves were linear over the concentration ranges, 0.5–2.5 μg/ml (hypericin), 0.35–1.6 μg/ml (pseudohypericin) and 5–50 μg/ml (hyperforin). The intra-day coefficients of variation obtained for hypericin, pseudohypericin and hyperforin were 4.4%, 5.4%, and 2.8%, respectively; inter-day CVs were 5.8%, 4.9%, and 2.5%, respectively. This method may be applied for the routine standardization of St. John's Wort products against hyperforin and the hypericins, the putative antidepressant principles in the herbal.     

Evaluation of Human Liver Slices and Reporter Gene Assays as Systems for Predicting the Cytochrome P450 Induction Potential of Drugs in Vivo in Humans

Purpose The aim of the study was to investigate the feasibility of predicting human in vivo cytochrome P450 (CYP) induction properties of drugs using in vitro methods. Methods The CYP induction potential of compounds was tested in human liver slices and in reporter gene assays for the aryl hydrocarbon receptor (AhR) and the pregnane X receptor (PXR). Results In human liver slices, CYP activities decreased dramatically over the experimental period, whereas mRNA levels could reliably be used to investigate CYP1A, 2C9, and 3A4 induction. However, the interindividual variations and demanding experimentation limit the use of liver slices in screening programs. Reporter gene assays are robust and reliable assays, amenable to high throughput screening. Several compounds activated AhR. The relevance of this activation, however, needs to be further investigated since there are no clear reports on drugs inducing CYP1A in vivo. The results from the PXR assay could be used to correctly classify compounds with known CYP3A induction properties when relating in vivo AUC_tot to PXR EC₅₀ values. Conclusions Liver slices are a valuable model to study the regulation of a larger number of enzymes by single compounds. The PXR reporter gene assay could be used as a reliable screening method to predict CYP3A induction in vivo.     

The influence of antioxidants on cigarette smoke-induced DNA single-strand breaks in mouse organs: a preliminary study with the alkaline single cell gel electrophoresis assay.

According to published information, the lung is the only clear target organ for tumors when mice are exposed to cigarette smoke. Liver, skin, and upper digestive tract are target organs when orally or dermally exposed to cigarette smoke condensate, but not kidney, brain, or bone marrow. We tested the genotoxicity of cigarette smoke in the known target organ (lung), possible target organs (stomach and liver), and non-target organs (kidney, brain, and bone marrow) of the mouse using the alkaline single-cell gel electrophoresis (SCG, or comet) assay, as modified by us. We also tested the effect of free radical scavengers on the genotoxicity of the smoke. Male ICR mice were exposed to cigarette smoke. DNA single-strand breaks (SSB) were measured by the SCG assay 15, 30, 60, 120, and 240 min after the exposure. Fifteen min after the animals were exposed for 1 min to a 6-fold dilution of smoke, SSB appeared in the lungs, stomach, and liver; the damage in the lungs and liver returned to almost control levels by 60 min, and that of the stomach by 120 min. Kidney, brain, and bone marrow DNA were not damaged. Exposure to more dilute smoke (12- or 24-fold dilution) did not cause DNA damage. Single oral pretreatment (100 mg/kg) of either ascorbic acid (VC) or alpha-tocopherol acetate (VE) 1 h before smoke inhalation prevented SSB in the stomach and liver, while VE but not VC significantly reduced SSB in the lung. Five consecutive days of either VC or VE (100 mg/kg/day) pretreatment completely prevented SSB in the lung, stomach, and liver. Thus, the SCG assay detected DNA SSB, induced by cigarette smoke, in the known target organ, two possible target organs, and none of the non-target organs. Antioxidants could prevent those effects, suggesting that free radicals may have been a source of the damage. Our results suggest the importance of the SCG assay as a tool in the study of genotoxicity and carcinogenicity.     

The use of d-amphetamine pellet implantation as a model for d-amphetamine tolerance in the mouse

The use of d-amphetamine pellet implantation as a method for producing rapid central drug tolerance was investigated. Mice were implanted with d-amphetamine pellets containing 2 mg of drug and were challenged 24 h later, a time when no detectable drug was present, with various doses of d-amphetamine i.p. Implantation was found to potentiate the stereotyped activity and produced tolerance to the exploratory activity induced by d-amphetamine. Daily pellet implantation for 3 days was not found to produce tolerance to the stereotyped activities. Animals administered a single pellet showed no difference in the brain disposition or metabolism of a subsequent dose of ³H-d-amphetamine. Twenty-four hour pellet implantation markedly increased the rate of conversion of ³H-tyrosine to ³H-dopamine (330%) and ³H-norepinephrine (61%) in the subcortex. However, this effect was reversed by the administration of 10 mg/kg of d-amphetamine.This work represents a portion of the dissertation submitted to the College of Pharmacy, Wayne State University, by R. J. Hitzemann in partial fulfillment of the requirements for the degree of Master of Science.This work was supported in part by USPHS Grants MH-11846 (EFD) and MH-20559 (HHL).