Prospective analysis of the outcome of subpectoral augmentation.
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Body Image↗
Biomedical subjects
E Hodgson
Publications and source records attributed to E Hodgson.
Pesticide metabolism in humans, including polymorphisms.
Recent epidemiologic studies involving Gulf War veterans or agricultural workers suggest that pesticide-pesticide or pesticide-drug interactions may be related to Gulf-War-related illnesses or elevated cancer risks, respectively. Metabolic interactions are one of many potential mechanisms requiring exploration in humans. The goal of the studies is to characterize important metabolic profiles of selected pesticides and examine potential interactions to characterize human risks associated with exposure. Pesticides examined using human liver microsomes and cytosolic fractions included chlorpyrifos, carbaryl and permethrin. The metabolic pathways involved include cytochrome P450 monooxygenases (CYP), esterases, and alcohol and aldehyde dehydrogenases. Specific isoforms and some polymorphic enzymes were characterized. Pesticide-pesticide interactions with metabolizing enzymes were demonstrated. Exposure of human hepatocytes to chlorpyrifos and permethrin demonstrated their potential to induce CYP isoforms using the bDNA (branched deoxyribonucleic acid) assay [used to monitor mRNA (messenger ribonucleic acid) levels]. These studies suggest that knowledge of human metabolic pathways will provide information that can aid the risk assessment process.
Environmental Exposure↗
Liquid based cytology: examination of its potential in a chlamydia screening programme.
OBJECTIVE: To assess the feasibility of testing for chlamydia directly on a single liquid based specimen (ThinPrep test) collected for cervical screening. METHOD: Cervical smears were taken using a Cervex spatula and rinsed in the liquid based cytology collection vial. Following this, the conventional sample for chlamydia testing was taken from the endocervix using an Abbott Collection kit. Cytological specimens were prepared using an automated slide processor. Residual cellular material and the conventional samples were sent to the laboratory where both were tested for chlamydia by ligase chain reaction (LCR). The manufacturer's protocol for LCR urine testing was modified to substitute 1 ml of PreservCyt suspension. RESULTS: 581 women had both swab and cytology suspension tested for Chlamydia trachomatis with LCR. There were 19 concordant positive and 562 concordant negative reports. The stability of chlamydia in the cytology suspension was maintained for at least 5 months. CONCLUSION: The findings lead us to conclude that samples collected for liquid based cytology using the ThinPrep test collection vial provide a potential platform for chlamydia screening, though the study established several issues to be addressed to make this a practical proposition.
Adult↗
The unforgettable finger tourniquet.
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Color↗
In vitro human phase I metabolism of xenobiotics I: pesticides and related chemicals used in agriculture and public health, September 2001.
Human phase I enzymes and their isoforms that metabolize pesticides are listed in a database that will be updated periodically. This initial version includes enzymes and isoforms that metabolize organophosphorus insecticides, chloroacetamide herbicides and triazine herbicides.
Cytochromes↗
Cloning, sequencing, heterologous expression, and characterization of murine cytochrome P450 3a25*(Cyp3a25), a testosterone 6beta-hydroxylase.
A full-length cDNA clone encoding a novel form of the cytochrome P450 3A subfamily (Cyp3a-25) has been isolated from a mouse liver cDNA library. The sequence contained 2010 base pairs and encoded a protein with 503 amino acids. The amino acid sequence shared greater identities with rat CYP3A18 (90%) and golden hamster CYP3A10 (81%) sequences than with known mouse sequences (Cyp3a-11, Cyp3a-13, Cyp3a-16, and Cyp3a-41 [68--70%]). CYP3A25 was expressed in the Escherichia coli PCWori(+) expression vector following slight modifications of the N- and C-terminals of the cDNA. The purified CYP3A25 was recognized on an immunoblot by CYP3A1 antibody and has a molecular weight of 50 kD. CYP3A25 was catalytically active in the 6 beta-hydroxylation of testosterone and the N-demethylation of benzphetamine and erythromycin. It was demonstrated by RT-PCR that the CYP3A25 mRNA is present in both fetal and adult tissues, including liver, lung, intestines, kidney, and brain. Northern blotting demonstrated that expression is greatest in the liver and small intestine.
Aging↗
Subunit compositions of crustacean haemocyanins are species-specific: evidence from non-decapod species.
Electrophoretic examination of dissociated haemocyanin subunits from a number of amphipod, decapod and isopod crustaceans supports the hypothesis that subunit composition is species-specific, despite marked within-species variation in many species. General patterns of heterogeneity on native PAGE gels were also evident between groupings within the Amphipoda. Gammarid amphipods could be split into two groups; one characterised by a high degree of heterogeneity and the other by a low degree of heterogeneity. The talitrid amphipods generally displayed a low degree of heterogeneity similar to, although still distinct from, the second gammarid category. Haemocyanin from the Hyalidae, a family allied to the talitrids was highly heterogeneous, similar to the first gammarid group and unlike the talitrids. Isopod haemocyanin banding patterns were more similar to one another than to any of the amphipod or decapod species examined. In general, the molecular weights of the amphipod Hcs tended to be greater than those of the isopods, with the decapods being lowest of all. It is suggested that Hc subunit heterogeneity may be a useful tool for investigating speciation and speciation events, and for reliably separating very closely-related species (e.g. Gammarus spp.), purely on the basis of their Hc subunit compositions.
Animals↗
Genetically modified plants and human health risks: Can additional research reduce uncertainties and increase public confidence?
So long as the risks to human health from transgenic plants remain potential rather than actual, and, in any event, appear lower than those from traditional plant breeding, hazard assessment need not be extensive. However, in view of current public attitudes to transgenic plants, it is necessary that those tests that are required, be based on logic, on sound science, and in accordance with the best scientific methodology. This is particularly the case with testing for food allergenicity. Current testing is largely indirect and based on comparisons with other known food allergens. Development of direct tests that involve interaction between the actual transgenic protein in question and the immune system is essential if confidence in the regulatory system is to be restored.
Food Hypersensitivity↗
Metabolism of chlorpyrifos by human cytochrome P450 isoforms and human, mouse, and rat liver microsomes.
One of the factors determining the toxicity of chlorpyrifos (CPS), an organophosphorus (OP) insecticide, is its biotransformation. CPS can be activated by cytochrome P450 (CYP) through a desulfuration reaction to form chlorpyrifos-oxon (CPO), a potent anticholinesterase. CPS can also be detoxified by CYP through a dearylation reaction. Using pooled human liver microsomes (HLM), a K(m(app)) of 30.2 microM and V(max(app)) of 0.4 nmol/min/mg of protein was obtained for desulfuration, and a K(m(app)) of 14.2 microM and a V(max(app)) of 0.7 nmol/min/mg of protein was obtained for dearylation. These activities are lower than those obtained from rat liver microsomes. Gender differences in humans were also observed with female HLM possessing greater activity than male HLM. Use of human CYP isoforms expressed in human lymphoblastoma cells demonstrated that CYP1A2, 2B6, 2C9*1, 2C19, and 3A4 are involved in CPS metabolism. CYP2B6 has the highest desulfuration activity, whereas dearylation activity is highest for 2C19. CYP3A4 has high activity for both dearylation and desulfuration. The use of phenotyped individual HLM demonstrated that predictions of metabolic activation and/or detoxication could be made based on relative amounts of CYP2B6, 2C19, and 3A4 in the microsomes. Thus, individuals with high CYP2C19 but low 3A4 and 2B6 are more active in dearylation than in desulfuration. Similarly, individuals possessing high levels of CYP2B6 and 3A4 have the greatest potential to form the activation product. These differences between individuals suggest that differential sensitivities to CPS may exist in the human population.
Animals↗
Identification of variants of CYP3A4 and characterization of their abilities to metabolize testosterone and chlorpyrifos.
CYP3A4 is the most abundant isoform of cytochrome P450 (CYP) in adult human liver. It metabolizes numerous clinically, physiologically, and toxicologically important compounds. The expression of CYP3A4 varies 40-fold in individual human livers, and metabolism of CYP3A4 substrates varies at least 10-fold in vivo. Single nucleotide polymorphisms (SNPs) in CYP3A4 were identified by direct sequencing of genomic DNA in 72 individuals from three different ethnic groups, including Caucasians, Blacks (African-Americans and African pygmies), and Asians. A total of 28 SNPs were identified, including five which produced coding changes M445T (CYP3A4*3), R162Q (CYP3A4*15), F189S (CYP3A4*17), L293P (CYP3A4*18), and P467S (CYP3A4*19). The latter four represent new alleic variants. Racial variability was observed for the frequency of individual SNPs. CYP3A R162Q was identified only in Black populations with an allelic frequency of 4%. CYP3A4 F189S and CYP3A4 M445T were identified in Caucasians with allelic frequencies 2% and 4%, respectively. L293P and P467S were only observed in Asians at allelic frequencies of 2%. The cDNAs for the F189S, L293P, M445T, and P467S mutant alleles were constructed by site-directed mutagenesis and expressed in an Escherichia coli expression system. Testosterone and the insecticide chlorpyrifos were used to assess the catalytic activities of the most common CYP3A4 allele (CYP3A4*1) and its allelic variants. CYP3A4 F189S exhibited lower turnover numbers for testosterone and chlorpyrifos, while CYP3A4 L293P had higher turnover numbers for both substrates. The turnover numbers of the CYP3A4 M445T and P467S alleles to metabolize these compounds were not significantly different from those of wild-type CYP3A4.
Alleles↗
Flavin-containing monooxygenase isoform specificity for the N-oxidation of tamoxifen determined by product measurement and NADPH oxidation.
The Km value for tamoxifen is 1.2 mM for mouse FMO1 (human FMO1 is not expressed in adults) and 1.4 mM for human FMO3, with no detectable activity being expressed toward tamoxifen by FMO5 from either mouse or human. These data are derived from experiments using 3H-tamoxifen as substrate in which the product, tamoxifen N-oxide, was measured directly. It was not possible to derive meaningful data from the measurement of NADPH consumption because Escherichia coli preparations, in the presence of tamoxifen, regardless of whether the E. coli was expressing an FMO isoform, consumed large amounts of NADPH without the appearance of tamoxifen N-oxide or other discernable product.
Animals↗
Comparative metabolism of chloroacetamide herbicides and selected metabolites in human and rat liver microsomes.
Acetochlor [2-chloro-N-(ethoxymethyl)-N-(2-ethyl-6-methyl-phenyl)-acetamide], alachlor [N-(methoxymethyl)-2-chloro-N-(2, 6-diethyl-phenyl)acetamide], butachlor [N-(butoxymethyl)-2-chloro-N-(2,6-diethyl-phenyl)acetamide], and metolachlor [2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl) acetamide] are pre-emergent herbicides used in the production of agricultural crops. These herbicides are carcinogenic in rats: acetochlor and alachlor cause tumors in the nasal turbinates, butachlor causes stomach tumors, and metolachlor causes liver tumors. It has been suggested that the carcinogenicity of these compounds involves a complex metabolic activation pathway leading to a DNA-reactive dialkylbenzoquinone imine. Important intermediates in this pathway are 2-chloro-N-(2,6-diethylphenyl)acetamide (CDEPA) produced from alachlor and butachlor and 2-chloro-N-(2-methyl-6-ethylphenyl)acetamide (CMEPA) produced from acetochlor and metolachlor. Subsequent metabolism of CDEPA and CMEPA produces 2,6-diethylaniline (DEA) and 2-methyl-6-ethylaniline (MEA), which are bioactivated through para-hydroxylation and subsequent oxidation to the proposed carcinogenic product dialkylbenzoquinone imine. The current study extends our earlier studies with alachlor and demonstrates that rat liver microsomes metabolize acetochlor and metolachlor to CMEPA (0.065 nmol/min/mg and 0.0133 nmol/min/mg, respectively), whereas human liver microsomes can metabolize only acetochlor to CMEPA (0.023 nmol/min/mg). Butachlor is metabolized to CDEPA to a much greater extent by rat liver microsomes (0.045 nmol/min/mg) than by human liver microsomes (< 0.001 nmol/min/mg). We have determined that both rat and human livers metabolize both CMEPA to MEA (0.308 nmol/min/mg and 0.541 nmol/min/mg, respectively) and CDEPA to DEA (0.350 nmol/min/mg and 0.841 nmol/min/mg, respectively). We have shown that both rat and human liver microsomes metabolize MEA (0.035 nmol/min/mg and 0.069 nmol/min/mg, respectively) and DEA (0.041 nmol/min/mg and 0.040 nmol/min/mg, respectively). We have also shown that the cytochrome P450 isoforms responsible for human metabolism of acetochlor, butachlor, and metolachlor are CYP3A4 and CYP2B6.
Acetamides↗
In vitro metabolism of alachlor by human liver microsomes and human cytochrome P450 isoforms.
Alachlor (2-chloro-N-methoxymethyl-N-(2,6-diethylphenyl)acetamide) is a widely used pre-emergent chloroacetanilide herbicide which has been classified by the USEPA as a probable human carcinogen. The putative carcinogenic metabolite, 2,6-diethylbenzoquinone imine (DEBQI), is formed through a complex series of oxidative and non-oxidative steps which have been characterized in rats, mice, and monkeys but not in humans. A key metabolite leading to the formation of DEBQI is 2-chloro-N-(2,6-diethylphenyl)acetamide (CDEPA). This study demonstrates that male human liver microsomes are able to metabolize alachlor to CDEPA. The rate of CDEPA formation for human liver microsomes (0.0031 +/- 0.0007 nmol/min per mg) is significantly less than the rates of CDEPA formation for rat liver microsomes (0.0353+/-0.0036 nmol/min per mg) or mouse liver microsomes (0.0106 +/- 0.0007). Further, we have screened human cytochrome P450 isoforms 1A1, 1A2, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, and 3A4 and determined that human CYP 3A4 is responsible for metabolism of alachlor to CDEPA. Further work is necessary to determine the extent to which humans are able to metabolize CDEPA through subsequent metabolic steps leading to the formation of DEBQI.
Acetamides↗
Constitutive expression and induction of CYP1B1 mRNA in the mouse.
Previous studies appeared to indicate that CYP1B1 was not constitutively expressed in mouse liver. In our laboratory, we demonstrated using aromatic hydrocarbon-responsive receptor knock-out (AHR-(-)/-) mice that both piperonyl butoxide (PBO) and acenaphtyhlene (ACN) are AHR-independent inducers of murine CYP1A2 and CYP1B1 mRNA. In the current study, we demonstrate both constitutive levels and induction of CYP1B1 in mouse liver. The induction of CYP1B1 mRNA by PBO or ACN was higher in DBA/2 (Ahrd) than in C57BL/6 (Ahrb-1) mice, while 3-methylcholanthrene induced CYP1B1 more in C57BL/6 than in DBA/2 mice. These results suggest that CYP1B1 may also be induced by more than one mechanism. In addition, constitutive expression of CYP1B1 was detected in liver, kidney, and lung of untreated C57BL/6 mice. There was no gender difference in CYP1B1 expression; however, in C57BL/6 mice, the kidney contained less CYP1B1 than either liver or lung.
Acenaphthenes↗
The orphan human pregnane X receptor mediates the transcriptional activation of CYP3A4 by rifampicin through a distal enhancer module.
Cytochrome P-450 3A4 (CYP3A4), the predominant cytochrome P-450 expressed in adult human liver, is subject to transcriptional induction by a variety of structurally unrelated xenobiotics, including the antibiotic rifampicin. The molecular mechanisms underlying this phenomenon are poorly understood. We transfected a human liver-derived cell line (HepG2) with various CYP3A4-luciferase reporter gene constructs containing a nested set of 5'-deletions of the CYP3A4 5'-flanking region. Rifampicin-inducible transcription of the reporter gene was observed only with the longest construct, which encompassed bases -13000 to +53 of CYP3A4 (3-fold induction). The responsive region was functional regardless of its position or orientation relative to the proximal promoter of CYP3A4 and was capable of conferring rifampicin-inducible expression on a heterologous promoter. Further deletion mutants localized the induction to bases -7836 to -7607. In vitro DNase I footprint analysis of this region revealed four protected sites (FP1, FP2, FP3, and FP4). Two of these sites, FP3 (bases -7738 to -7715) and FP4 (bases -7698 to -7682), overlapped binding motifs for the orphan human pregnane X receptor (hPXR). Cotransfection of responsive constructs with a hPXR expression vector substantially increased the rifampicin-inducibility to approximately 50-fold. In addition, the rifampicin-responsive constructs were strongly activated by a range of CYP3A inducers. Finally, we demonstrate cooperativity between elements within the distal enhancer region and cis-acting elements in the proximal promoter of CYP3A4. Our results provide evidence for the existence of a potent enhancer module, 8 kb distal to the transcription start point, which mediates the transcriptional induction of CYP3A4 by activators of hPXR.
3T3 Cells↗
Cloning and expression of murine CYP2Cs and their ability to metabolize arachidonic acid.
Five murine cytochrome P450 (CYP) 2C cDNAs were cloned and characterized, including four new members of this subfamily: CYP2C37, CYP2C38, CYP2C39, and CYP2C40. The cDNAs ranged from 1716 to 1812 bp in length and encoded polypeptides of 490 amino acid residues except for CYP2C40, which contained an additional glutamic acid residue at the carboxyl terminus. The amino acid identity of the murine CYP2Cs ranged from 69 to 92%, while the overall amino acid identity was 60%; however, within the six putative substrate recognition sites the identity was only 25 to 41%, suggesting possible differences in substrate specificity and product profiles. The CYP2C cDNAs were expressed in Escherichia coli following modification of the N-terminus. All five recombinant CYP2Cs metabolized arachidonic acid, but with different metabolic profiles and catalytic rates. Based on coelution with authentic standards on reverse-phase HPLC, themajor metabolites were tentatively identified asfollows: CYP2C29 and CYP2C39 produced 14, 15-cis-epoxyeicosatrienoic acid (EET); CYP2C37 produced 12-hydroxyeicosatetraenoic acid (HETE); CYP2C38 produced 11,12-EET; and CYP2C40 produced an unidentified metabolite that coeluted with 16-,17-, and 18-HETEs. The turnover numbers for CYP2C29, CYP2C37, CYP2C38, CYP2C39, and CYP2C40 were 0.34, 1.12, 5.15, 0.51, and 0.15 nmol/nmol/min, respectively. Reverse transcriptase-polymerase chain reaction demonstrated the presence of CYP2C29 mRNA in liver as well as in extrahepatic tissues including brain, kidney, lung, heart, and intestine. CYP2C38 and CYP2C40 were found in liver, brain, kidney, and intestine, with trace amounts in lung and heart, while CYP2C37 and CYP2C39 appeared to be liver specific.
Amino Acid Sequence↗
Effect of metformin on bile salt circulation and intestinal motility in type 2 diabetes mellitus.
Gastrointestinal symptoms can be a limiting factor in optimizing metformin therapy, particularly at the onset of treatment. The underlying cause remains unclear. We have investigated whether metformin changes oral-caecal transit and if it causes bile salt malabsorption using the lactulose breath test and orally administered 14C-glycocholate followed by breath 14CO2 measurement over 6 h and stool collection for 72 h, respectively. Twenty-four diet and/or sulphonylurea treated patients underwent 7 days of baseline investigations before entering a randomized double-blind crossover study of 21 days duration with either metformin (850 mg bd) or placebo. No difference was observed in the oral-caecal transit time but a change in fasting plasma glucose was observed of 2.6 mmol l-1 (95% CI 1.3, 3.8). Significant increases in percentage 14CO2 breath elimination were observed during treatment with metformin (9.7 +/- 6.3) compared with placebo (3.1 +/- 1.9) p = 0.020. In addition, percentage faecal 14C bile salt excretion was increased with metformin (17.2 +/- 9.9 vs 10.1 +/- 6.9) p = 0.037. A significant association (p = 0.002) emerged for stool bile salt content and liquidity of the stool. We conclude that metformin may cause gastrointestinal disturbances by reducing ileal bile salt reabsorption leading to elevated colonic bile salt concentrations.
Adult↗
Molecular cloning, sequence, and expression of mouse flavin-containing monooxygenases 1 and 5 (FMO1 and FMO5).
Full-length cDNA clones encoding FMO1 and FMO5 have been isolated from a library constructed with mRNA from the liver of a female CD-1 mouse. The derived sequence of FMO1 contains 2310 bases: 1596 in the coding region, 301 in the 5'-flanking region, and 413 in the 3'-flanking region. The sequence for FMO5 consists of 3168 bases; 1599 in the coding region, 812 in the 5'-flanking region, and 757 in the 3'-flanking region. The sequence of FMO1 encodes a protein of 532 amino acids with a predicted molecular weight of 59.9 kDa and shows 83.3% identity to human FMO1 and 83-94% identity to other FMO1 homologs. FMO5 encodes a protein of 533 amino acids with a predicted molecular weight of 60.0 kDa and 84.1% identity to human FMO5 and 83-84% identity to other FMO5 orthologs. Two GxGxxG putative pyrophosphate binding domains exist beginning at positions 9 and 191 for FMO1, and 10 and 192 for FMO5. Mouse FMO1 and FMO5 were expressed in E. coli and show similar mobility to the native proteins as determined by SDS-PAGE. The expressed FMO1 protein showed activity toward methimazole, and FMO5 was active toward noctylamine. In addition, FMO1 was shown to metabolize radiolabeled phorate, whereas FMO5 showed no activity toward phorate.
Animals↗