Biomedical subjects

D R Doerge

Publications and source records attributed to D R Doerge.

At least 19 recordsLinked to original sources

PubMed2003

Effect of dietary genistein on cell replication indices in C57BL6 mice.

The phytoestrogen and isoflavone, genistein, inhibited the activity of the DNA synthesis-related enzyme, topoisomerase-II (topo-II), altered cell-cycle traverse and produced cell death in cell culture models. In order to examine the potential effects of genistein on cell replication and cell death in an animal model, 8-week-old C57BL6 mice were fed either a control diet or one containing one of five doses (100-2000 ppm) of genistein for 28 days. At the end of the feeding period, both male and female mice were sacrificed and the serum isoflavone and aglycone levels determined by liquid chromatography with electrospray tandem mass spectrometry (LC-ES/MS/MS). Immunohistochemistry was utilized to measure the cell replication and cell death rates in the small intestine. Total isoflavone concentration increased from below the limits of detection (0.001 microM) in control animals to 0.28 microM in male and 0.54 microM in female mice fed the 2000 ppm diet. A decrease in the percentage of cells in G(0) and an increase in the percentage of cells in S-phase, consistent with topo-II-induced S-phase arrest, was found in the duodenum and jejunum of the small intestine. Thus, genistein appears to accumulate to a sufficient level to affect topo-II activity in the small intestine.

Administration, Oral↗

PubMed2002

Analysis of DNA adducts from chemical carcinogens and lipid peroxidation using liquid chromatography and electrospray mass spectrometry.

The identification and dosimetry of DNA adducts are cornerstones of research on cancer etiology in experimental animals and humans. DNA adducts can result from exposure to exogenous chemical carcinogens or through reactions with endogenous by-products of oxidative metabolism. An important research need is high throughput methodology for quantification of any and all adducts that are present at trace amounts in DNA derived from target tissues of animals and humans. This review describes some recent progress made through applications of liquid chromatography coupled with mass spectrometry to structural characterization of unknown DNA adducts and highly sensitive quantitative analysis of target adducts.

Animals↗

PubMed2001

Degradation of biphenyl by Mycobacterium sp. strain PYR-1.

The metabolism of biphenyl by Mycobacterium sp. PYR-1 was investigated. The Mycobacterium sp. degraded >98% of the biphenyl added within 72 h. Analysis of ethyl acetate extracts of the culture medium by HPLC indicated that benzoic acid was the major metabolite. Other products were 4-hydroxybiphenyl, 4-hydroxybenzoic acid, and 5-oxo-5-phenylpentanoic acid. The metabolites were characterized by mass and 1H NMR spectrometry. Identification of benzoic acid and 5-oxo-5-phenylpentanoic acid indicates that biphenyl degradation by Mycobacterium sp. PYR-1 is generally similar to known pathways. A novel alternative metabolic pathway consisted of monooxygenation at C-4 of biphenyl to give 4-hydroxybiphenyl, with subsequent degradation via ring cleavage to 4-hydroxybenzoic acid.

Benzoic Acid↗

PubMed2001

Effect of soy isoflavone supplementation on markers of oxidative stress in men and women.

Dietary intake of soy has been linked with decreased cancer risk, and the active compounds in soy that have been identified include the isoflavones genistein and daidzein. Since these compounds have antioxidant properties, we examined levels of oxidative damage in blood of six women and six men before and during soy supplementation using Novasoy tablets. Blood samples were obtained at weekly intervals for 3 weeks from the women taking 50-mg isoflavones once daily and the men taking 50-mg isoflavones twice daily. Plasma levels of genistein and daidzein increased after supplementation with maximal levels occurring at 2 weeks for the women while levels in men kept increasing over the 3 weeks of study. There was wide variation between individuals in the levels of isoflavones achieved. Mean levels of 5-hydroxymethyl-2'-deoxyuridine (5-OHmdU) in DNA from nucleated blood cells decreased after 1 week of supplementation in the women, with a decrease of 47% in mean 5-OHmdU levels after 3 weeks. In men, mean 5-OHmdU levels did not decrease until after 3 weeks of supplementation, at which there was 61% decrease. Mean plasma levels of 8-isoprostanes were not changed appreciably in either men or women. These pilot results suggest that soy isoflavone supplementation decreases levels of oxidative DNA damage in humans, and this may be a mechanism behind the cancer-preventive effects of soy isoflavones.

Adult↗

PubMed2001

Metabolism of anthracene by a Rhodococcus species.

A Rhodococcus sp. isolated from contaminated river sediment was investigated to determine if the isolate could degrade high molecular mass polycyclic aromatic hydrocarbons. The Rhodococcus sp. was able to utilize anthracene (53%), phenanthrene (31%), pyrene (13%), and fluoranthene (5%) as sole source of carbon and energy, but not naphthalene or chrysene. In a study of the degradation of anthracene by a Rhodococcus sp., the identification of ring-fission products indicated at least two ring-cleavage pathways. One results in the production of 6,7-benzocoumarin, previously shown to be produced chemically from the product of meta cleavage of 1,2-dihydroxyanthracene, a pathway which has been well established in Gram-negative bacteria. The second is an ortho cleavage of 1,2-dihydroxyanthracene that produces 3-(2-carboxyvinyl)naphthalene-2-carboxylic acid, a dicarboxylic acid ring-fission product. This represents a novel metabolic pathway only identified in Gram-positive bacteria.

Anthracenes↗

PubMed2001

Quantitative analysis of etheno-2'-deoxycytidine DNA adducts using on-line immunoaffinity chromatography coupled with LC/ES-MS/MS detection.

Etheno DNA adducts, including 3,N4-etheno-2'-deoxycytidine (etheno-dC), are promutagenic lesions present in normal animal and human tissues. These DNA adducts are believed to be important in the etiology of cancer. Existing methods for quantifying etheno-dC use 32p. postlabeling. Although highly sensitive, postlabeling requires the use of an energetic radioisotope and considerable time and effort. The new methodology reported here permits automated quantification of trace levels of etheno-dC in crude DNA hydrolysates on the order of 5 adducts in 10(8) normal nucleotides from 100-microg samples of DNA. This was accomplished by using on-line immunoaffinity chromatography, a reverse-phase LC separation on graphitized carbon, tandem mass spectrometric detection, and an isotopically labeled internal standard. The automated procedures permitted analysis of 4 DNA hydrolysates/hr. The sensitivity using immunoaffinity cleanup was approximately 100-fold greater than that observed when using a silica-based trapping system. The validated method was applied to the analysis of etheno-dC in commercial calf thymus DNA, untreated mouse liver, and untreated rat liver DNA. The demonstrated level of performance suggests future applicability of this method in studies of cancer in humans and experimental animals.

Animals↗

PubMed2001

Development of a (32)P-postlabeling/HPLC method for detection of dehydroretronecine-derived DNA adducts in vivo and in vitro.

Pyrrolizidine alkaloids are naturally occurring genotoxic chemicals produced by a large number of plants. Metabolism of pyrrolizidine alkaloids in vivo and in vitro generates dehydroretronecine (DHR) as a common reactive metabolite. In this study, we report the development of a (32)P-postlabeling/HPLC method for detection of (i) two DHR-3'-dGMP and four DHR-3'-dAMP adducts and (ii) a set of eight DHR-derived DNA adducts in vitro and in vivo. The approach involves (1) synthesis of DHR-3'-dGMP, DHR-3'-dAMP, and DHR-3',5'-dG-bisphosphate standards and characterization of their structures by mass and (1)H NMR spectral analyses, (2) development of optimal conditions for enzymatic DNA digestion, adduct enrichment, and (32)P-postlabeling, and (3) development of optimal HPLC conditions. Using this methodology, we have detected eight DHR-derived DNA adducts, including the two epimeric DHR-3',5'-dG-bisphosphate adducts both in vitro and in vivo.

Animals↗

PubMed2001

Metabolic activation of the tumorigenic pyrrolizidine alkaloid, riddelliine, leading to DNA adduct formation in vivo.

Riddelliine is a representative naturally occurring genotoxic pyrrolizidine alkaloid. We have studied the mechanism by which riddelliine induces hepatocellular tumors in vivo. Metabolism of riddelliine by liver microsomes of F344 female rats generated riddelliine N-oxide and dehydroretronecine (DHR) as major metabolites. Metabolism was enhanced when liver microsomes from phenobarbital-treated rats were used. Metabolism in the presence of calf thymus DNA resulted in eight DNA adducts that were identical to those obtained from the reaction of DHR with calf thymus DNA. Two of these adducts were identified as DHR-modified 7-deoxyguanosin-N(2)-yl epimers (DHR-3'-dGMP); the other six were DHR-derived DNA adducts, but their structures were not characterized. A similar DNA adduct profile was detected in the livers of female F344 rats fed riddelliine, and a dose-response relationship was obtained for the level of the total (eight) DHR-derived DNA adducts and the level of the DHR-3'-dGMP adducts. These results suggest that riddelliine induces liver tumors in rats through a genotoxic mechanism and the eight DHR-derived DNA adducts are likely to contribute to liver tumor development.

Animals↗

PubMed2001

Physiological concentrations of dietary genistein dose-dependently stimulate growth of estrogen-dependent human breast cancer (MCF-7) tumors implanted in athymic nude mice.

Previously our laboratory has shown that the soy isoflavone, genistein, stimulates growth of human breast cancer (MCF-7) cells in vivo and in vitro. In this study, the dose-response analysis of genistein at the physiologically achievable concentration range between 125 and 1,000 microg/g in the diet was conducted in ovariectomized athymic nude mice implanted with MCF-7 cells. We hypothesized that genistein at this concentration range can stimulate dose-dependently the breast tumor growth, cell proliferation and an estrogen-responsive pS2 gene induction. Tumor size and body weight were monitored weekly. At completion of the study, we analyzed cellular proliferation of tumors using incorporation of BrdU, pS2 expression of tumors using a Northern blot analysis and total genistein level in plasma using liquid chromatography-isotope dilution mass spectrometry (LC-ES/MS). Dietary genistein (> or = 250 microg/g) increased tumor size in a dose-dependent manner [8.4x the negative control (NC) group in the 250 microg/g group, 12.0x in the 500 microg/g group, 20.2x in the 1,000 microg/g group and 23.2x in the positive control (PC) group]. The percentage of proliferating cells was significantly increased by genistein at and above 250 microg/g (5.3x the NC group in the 250 microg/g, 5.6x in the 500 microg/g, 5.0x in the 1,000 microg/g and 4.8x in the PC group). Expression of pS2 mRNA was also significantly increased with increasing dietary genistein levels (11.25x the NC group in the 500 microg/g group and 15.84x in the 1,000 microg/g group). Total plasma genistein concentrations were between 0.39 and 3.36 micromol/L in mice fed between 125 and 1,000 microg/g genistein. In conclusion, dietary treatment with genistein at physiological concentrations produces blood levels of genistein sufficient to stimulate estrogenic effects, such as breast tumor growth, cellular proliferation and pS2 expression in athymic mice in a dose-responsive manner similar to that seen in vitro.

Administration, Oral↗

PubMed2001

Degradation of phenanthrene and anthracene by cell suspensions of Mycobacterium sp. strain PYR-1.

Cultures of Mycobacterium sp. strain PYR-1 were dosed with anthracene or phenanthrene and after 14 days of incubation had degraded 92 and 90% of the added anthracene and phenanthrene, respectively. The metabolites were extracted and identified by UV-visible light absorption, high-pressure liquid chromatography retention times, mass spectrometry, (1)H and (13)C nuclear magnetic resonance spectrometry, and comparison to authentic compounds and literature data. Neutral-pH ethyl acetate extracts from anthracene-incubated cells showed four metabolites, identified as cis-1,2-dihydroxy-1,2-dihydroanthracene, 6,7-benzocoumarin, 1-methoxy-2-hydroxyanthracene, and 9,10-anthraquinone. A novel anthracene ring fission product was isolated from acidified culture media and was identified as 3-(2-carboxyvinyl)naphthalene-2-carboxylic acid. 6,7-Benzocoumarin was also found in that extract. When Mycobacterium sp. strain PYR-1 was grown in the presence of phenanthrene, three neutral metabolites were identified as cis- and trans-9,10-dihydroxy-9,10-dihydrophenanthrene and cis-3,4-dihydroxy-3,4-dihydrophenanthrene. Phenanthrene ring fission products, isolated from acid extracts, were identified as 2,2'-diphenic acid, 1-hydroxynaphthoic acid, and phthalic acid. The data point to the existence, next to already known routes for both gram-negative and gram-positive bacteria, of alternative pathways that might be due to the presence of different dioxygenases or to a relaxed specificity of the same dioxygenase for initial attack on polycyclic aromatic hydrocarbons.

Anthracenes↗

PubMed2001

Molecular cloning, nucleotide sequence, and expression of genes encoding a polycyclic aromatic ring dioxygenase from Mycobacterium sp. strain PYR-1.

Mycobacterium sp. strain PYR-1 degrades high-molecular-weight polycyclic hydrocarbons (PAHs) primarily through the introduction of both atoms of molecular oxygen by a dioxygenase. To clone the dioxygenase genes involved in PAH degradation, two-dimensional (2D) gel electrophoresis of PAH-induced proteins from cultures of Mycobacterium sp. strain PYR-1 was used to detect proteins that increased after phenanthrene, dibenzothiophene, and pyrene exposure. Comparison of proteins from induced and uninduced cultures on 2D gels indicated that at least six major proteins were expressed (105, 81, 52, 50, 43, and 13 kDa). The N-terminal sequence of the 50-kDa protein was similar to those of other dioxygenases. A digoxigenin-labeled oligonucleotide probe designed from this protein sequence was used to screen dioxygenase-positive clones from a genomic library of Mycobacterium sp. strain PYR-1. Three clones, each containing a 5,288-bp DNA insert with three genes of the dioxygenase system, were obtained. The genes in the DNA insert, from the 5' to the 3' direction, were a dehydrogenase, the dioxygenase small (beta)-subunit, and the dioxygenase large (alpha)-subunit genes, arranged in a sequence different from those of genes encoding other bacterial dioxygenase systems. Phylogenetic analysis showed that the large alpha subunit did not cluster with most of the known alpha-subunit sequences but rather with three newly described alpha subunits of dioxygenases from Rhodococcus spp. and Nocardioides spp. The genes from Mycobacterium sp. strain PYR-1 were subcloned and overexpressed in Escherichia coli with the pBAD/ThioFusion system. The functionality of the genes for PAH degradation was confirmed in a phagemid clone containing all three genes, as well as in plasmid subclones containing the two genes encoding the dioxygenase subunits.

Cloning, Molecular↗

PubMed2001

Biotransformation of malachite green by the fungus Cunninghamella elegans.

The filamentous fungus Cunninghamella elegans ATCC 36112 metabolized the triphenylmethane dye malachite green with a first-order rate constant of 0.029 micromol x h(-1) (mg of cells)(-1). Malachite green was enzymatically reduced to leucomalachite green and also converted to N-demethylated and N-oxidized metabolites, including primary and secondary arylamines. Inhibition studies suggested that the cytochrome P450 system mediated both the reduction and the N-demethylation reactions.

Biodegradation, Environmental↗

PubMed2000

Dietary genistein inactivates rat thyroid peroxidase in vivo without an apparent hypothyroid effect.

Biological effects of genistein are currently under investigation by the National Toxicology Program because of widespread and increasing soy consumption by humans and evidence for modulation of endocrine function. Rats were exposed to genistein aglycone in soy-free feed fortified at 0, 5, 100, and 500 ppm starting in utero through 20 weeks. Thyroid glands and serum were analyzed for total genistein (aglycone + conjugates) using HPLC with electrospray mass spectrometric detection. Microsomal thyroid peroxidase (TPO) activity was measured spectrophotometrically. The total genistein content in rat serum was as high as 8 microM, and significant dose-dependent increases of genistein in thyroid tissue up to 1 pmol/mg were found in male and female rats. The activity of TPO in male and female rats was found to be reduced by up to 80% in a dose-dependent manner. Male and female rats consuming a standard soy-based rodent diet (NIH 31) had TPO activity approximately 50% lower than rats consuming a soy-free diet and this loss was commensurate with measured serum levels of isoflavones. Suicide inactivation of rat, porcine, and human TPO was observed in vitro at concentrations of genistein aglycone comparable to those measured in rat thyroids. Thyroid hormone levels (T3, T4, TSH) in serum, thyroid weights, and histopathology showed no differences between treated and untreated groups. These findings suggest that, even though substantial amounts of TPO activity are lost concomitant to soy isoflavone consumption by normal rats, the remaining enzymatic activity is sufficient to maintain thyroid homeostasis in the absence of additional perturbations.

Animals↗

PubMed2000

DNA adduct formation by Fusarium culture extracts: lack of role of fusarin C.

Fusarium fungi have been shown to infect corn and other crops worldwide, and have a significant impact on human health through loss of crops or contamination of food with mycotoxins. Isolates of Fusarium fungi from an area of South Africa with high incidence of esophageal cancer have been shown to induce esophageal and liver cancer in rats. Several isolates of Fusarium fungi were grown on corn to determine if genotoxic products were produced. We report the incubation of methanol extracts of Fusarium verticillioides cultures with DNA in the presence of rat liver fractions (S9) resulted in the formation of a unique DNA adduct that was detected by (32)P-postlabeling. Fusarin C was purified from cultures of Fusarium verticillioides RRC 415, and was not responsible for the formation of the DNA adduct. Treatment of the methanolic extracts with ultraviolet B radiation reduced the fusarin C content in the extract; however, this had no effect on the formation of the DNA adduct following incubation of the extract with DNA and S9. The unique DNA adduct was formed following the incubation of several Fusarium verticillioides isolates from the US and South Africa, while extracts of cultures of Fusarium graminearium and Fusarium sacchari isolates formed very little of the DNA adduct when incubated with DNA and S9. These data suggest that neither fusarin C nor any of its metabolites are responsible for formation of the DNA adduct, and that an unidentified compound is present in F. verticillioides cultures that forms a DNA adduct, and may be important in the etiology of human esophageal cancer.

Animals↗

PubMed2000

Quantitative analysis of interindividual variation of glutathione S-transferase expression in human pancreas and the ambiguity of correlating genotype with phenotype.

Analysis of glutathione S-transferases (GSTs) of the alpha, mu, and pi classes by reverse-phase high-performance liquid chromatography and electrospray-ionization mass spectrometry in 43 samples of normal human pancreas demonstrated a wide variation in expression of subunits P1, A1, A2, A4, M1, M2, and M3 and the presence of a novel form designated GST "A5." GSTA2 consisted of three forms that were differentially expressed between individuals in a manner consistent with allelic polymorphism at the hGSTA2 locus. Expression, in terms of microg GST subunit/mg cytosolic protein, varied by 6-15-fold for subunits P1, A2, and M3 and 17-30-fold in the case of GSTs A1 and M2. Less consistently expressed were GSTs M1a, M1b, A4, and A5. Among these, GSTM1 expression (excluding M1-null samples) varied 12-fold between samples, whereas GST A4 and A5 expression varied approximately 50-100-fold between samples, well beyond the range of other subunits, suggesting that their expression is highly inducible. Linear correlations (P < 0.001-0.003) existed between levels of the most consistently expressed GST, GSTP1, and total GSTs, GSTA2 and M3, and in GSTM1-positive samples, between GSTM1, M3, and P1. The correlation between GST subunits P1 and M3 was bimodal according to M1 genotype, reflecting the presence of the regulatory element in hGSTM3*B that is linked with the hGSTM1*A genotype. It is concluded that although a degree of regulation of expression of GSTs occurs in human pancreas, the variability of phenotype is high and might obscure the effects of genetic polymorphisms on individual cancer susceptibility. Interindividual variation of GST expression is, therefore, a factor that should be taken account of in epidemiological studies.

Adolescent↗

PubMed2000

Analysis of erythromycin by liquid chromatography/mass spectrometry using involatile mobile phases with a novel atmospheric pressure ionization source.

A critical limitation of electrospray ionization (ESI) liquid chromatography/mass spectrometry (LC/MS) sources is the susceptibility to blockage of interface orifices due to the deposition of involatile components from the sample and/or mobile phase. These components, including salts, buffers, and ion-pairing agents, can be essential to the performance of the chosen analytical method. We report here the performance enhancements provided by a novel atmospheric pressure ionization (API) source in the analysis of erythromycin A (ERY) using mobile phases that contain involatile components. The enhanced robustness of the new source is derived from the use of a continuous flow of aqueous solvent at the sampling cone orifice that maintains unobstructed ion transmission. The ESI mass spectral responses measured for ERY, using an LC separation that incorporates 10 mM sodium phosphate with and without 10 mM octane sulfonate, were monitored by repeated injections over 13-15 h total analysis time. Minimal effects on ESI mass spectral responses (integrated peak area) or chromatographic performance (peak shape, retention time) were observed during these studies. In the absence of the aqueous cleaning flow, complete loss of mass spectral responses and total blocking of the sampling cone was observed in less than 30 min. Responses for ERY spiked into chicken and beef liver, and catfish muscle at or below the regulatory level of interest (100 ppb), were quantified by internal standard calibration using this procedure. These results demonstrate the ability of a novel API-MS ion source to perform analyses that require the use of involatile mobile phase additives.

Animals↗

PubMed2000

Analysis of methylphenidate and its metabolite ritalinic acid in monkey plasma by liquid chromatography/electrospray ionization mass spectrometry.

Methylphenidate (MP, Ritalin) is a psychotropic drug widely prescribed to children for treating the symptoms of attention deficit disorder with and without hyperactivity. Because little information exists about the effects of chronic MP administration on cognitive function in children, measures of behavior changes in non-human primates are important surrogates. An essential component of such studies is the determination of MP plasma levels under chronic and acute dosing conditions. An analytical method was developed that provided sufficient sensitivity to measure low levels of the active parent drug (lower limit of quantitation = 0.25 ng/mL) and the inactive metabolite, ritalinic acid (RA), in monkey plasma as well as the ability to conveniently analyze large numbers of samples. The method uses a polymeric reversed-phase sorbent for solid phase extraction, an efficient reverse-phase high performance liquid chromatography (HPLC) separation, deuterated internal standards for isotope dilution quantification of MP and RA, and detection by sensitive electrospray ionization mass spectrometry (ES-MS) with a single quadrupole instrument. The method responses are linear over the range of plasma concentrations of MP and RA observed in monkeys, gives respective analyte recoveries of 75 and 60% with reasonable precision and accuracy, and demonstrates robust MS performance for rapid determination of MP/RA plasma levels. The average peak MP concentration (ca. 16 ng/mL) and half-lives for MP and RA elimination in monkeys (1.79 and 2.31 h, respectively) were not significantly different under acute vs. chronic dosing conditions and were comparable to values previously reported from human studies.

Animals↗

PubMed2000

On-line sample preparation using restricted-access media in the analysis of the soy isoflavones, genistein and daidzein, in rat serum using liquid chromatography electrospray mass spectrometry.

Soy isoflavones are the subject of many investigations in experimental animals and humans regarding possible modulation of endocrine activity and chemoprevention of carcinogenesis. Genistein and daidzein, the principal biologically active isoflavones in soy, were measured using on-line solid-phase extraction (SPE) and liquid chromatography electrospray mass spectrometry (LC/ES-MS) detection in serum of rats consuming a common open-formula (NIH 31) chow that contained approximately 30 microg each of genistein and daidzein per gram of feed and a specially designed 'soy-free' chow that contained approximately 60-fold lower isoflavones. The use of a restricted-access/reverse phase trap cartridge and automated column switching permitted rapid and robust analytical performance with many injections of plasma onto a reverse phase LC column. Enzymatic deconjugation and a single centrifugation step were the only sample preparation steps required. The limit of detection for the isoflavones, based on the MS responses observed in serum from male and female rats consuming the soy-free chow, was 0.020 microM. The method, which uses deuterated isoflavones as internal standards, was determined to be accurate using spiked control serum (102-110% of added amounts) and precise using spiked control serum and incurred serum (<6% relative standard deviation). The average genistein and daidzein levels were determined in female (0.62 and 0.25 microM, respectively) and male rats (0.35 and 0.20 microM, respectively) consuming the standard diet. The sex difference observed for serum genistein concentrations was statistically significant (p < 0.0001). These results underscore the potential impact of standard open-formula diets on the results from rodent bioassays of biological activity.

Animals↗