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Biomedical subjects

D E Kelly

Publications and source records attributed to D E Kelly.

At least 55 records · Page 3Linked to original sources

Metabolism of organochlorine pesticides: the role of human cytochrome P450 3A4.

Organochlorine compounds are widely used as pesticides and are substantial environmental pollutants and carcinogens due to their extensive environmental release. In the present study biotransformation of these pesticides was observed in the microsomal fractions and whole cells of Saccharomyces cerevisiae expressing human cytochrome P450 3A4. In both in vitro and in vivo studies, hexachlorobenzene and pentachlorobenzene were metabolised into pentachlorophenol which was further transformed into tetrachlorohydroquinone. Metabolites were identified by thin layer chromatography and 13C-NMR spectroscopy. The formation of products was observed only in the presence NADPH in microsomal fractions and no activity was observed in control microsomal fractions, or in whole cells.

Aryl Hydrocarbon Hydroxylases↗

Studies on the toxicity of analogues of dapsone in-vitro using rat, human and heterologously expressed metabolizing systems.

Three metabolizing systems (rat, heterologously expressed CYP3A4 and human liver) were used to evaluate 12 analogues of dapsone (4,4'diaminodiphenylsulphone) in-vitro. Methaemoglobin formation in a two-compartment and cytotoxicity in a single-compartment model were studied using human erythrocytes and neutrophils, respectively, as target cells. In the two-compartment system using rat microsomes as a generating system and methaemoglobin as an endpoint, the least potent methaemoglobin formers tested were the 2-methyl-4-propylamino (AXDD14), 2-hydroxy-4-4'amino (ABDD5) derivatives and a sulphone/trimethoprim derivative (K-130). Dapsone itself, a 2-methoxy-4-ethylamino (W10) and a 2-hydroxyl-4-ethylamino compound (ABDD39) were the most toxic. In the single-compartment cytotoxicity test using rat microsomes, AXDD14 was again among the least toxic, as was a 2-methyl 4-cyclopentyl derivative (AXDD17) and surprisingly ABDD39. The most cytotoxic compounds again included dapsone itself as well as two 2-trifluoromethyl derivatives. The only significant methaemoglobin formation and cytotoxicity shown with the heterologously expressed human CYP 3A4 was with AXDD14, which was extensively activated. Interestingly, metabolism of dapsone was low using the expressed CYP 3A4. In the two-compartment system using human liver microsomes, AXDD14, K-130 and ABDD5 were oxidized to a significantly lesser extent compared with dapsone and these preliminary findings indicate that future development of these compounds may be worthwhile.

Animals↗

Purification and reconstitution of activity of Saccharomyces cerevisiae P450 61, a sterol delta 22-desaturase.

P450 was purified from microsomal fractions of a strain of Saccharomyces cerevisiae which contained detectable P450 despite the disruption of CYP51A1. The P450 had a molecular mass of 58 kDa, similar to P450 51A1, and in a reconstituted assay with rabbit NADPH-P450 reductase and dilauryl phosphotidylcholine exhibited activity for conversion of ergosta-5,7-dienol into ergosterol. N-Terminal amino acid sequencing of the purified protein corresponded to the translated sequence of P450 61 which was recently identified during sequencing of chromosome XIII. This allowed the function of this family of P450 to be identified as sterol delta 22-desaturation in the pathway of ergosterol biosynthesis.

Amino Acid Sequence↗

Mode of action and resistance to azole antifungals associated with the formation of 14 alpha-methylergosta-8,24(28)-dien-3 beta,6 alpha-diol.

Azole antifungal compounds inhibit sterol 14 alpha-demethylase. They are used extensively for the treatment of immunocompromised patients where fungal infection is common and often results in death. Resistance to the compounds is emerging, particularly in fungal pathogens obtained from AIDS patients undergoing prolonged therapy. We show here that cell growth arrest correlates with the accumulation of 14 alpha-methyl-ergosta-8,24(28)-dien-3 beta,6 alpha-diol in a yeast strain with a sterol 14 alpha-demethylase gene disruption, which mimics stringent treatment conditions. Cells can overcome the effect of such a block by a suppressor mutation in sterol delta 5,6 desaturation and acquire azole resistance. Plasmid-based complementation of sterol 14 alpha-demethylase defect does not alter the azole susceptibility of strains containing these suppressor mutations, showing resistance is due entirely to the delta 5.6 desaturase defect.

Antifungal Agents↗

Metabolism of the herbicide chlortoluron by human cytochrome P450 3A4.

Studies were carried out to investigate the metabolism of herbicide chlortoluron in the microsomal fractions and whole cells of Saccharomyces cerevisiae expressing human cytochrome P450 3A4. Both whole cells and microsomal fractions of yeast expressing human cytochrome P450 3A4 exhibited a typical dithionite-reduced, CO-difference absorbance spectrum with maximum absorbance at 448 nm. Chlortoluron produced a type I binding spectrum with cytochrome P450 3A4 with a Ks value of 200 microM. Chlortoluron was metabolised into four metabolites; hydroxylated-N-monodemethylated, hydroxylated ring methylated, N-didemethylated and N-monodemethylated products. Chlortoluron metabolism was absolutely dependent on NADPH and no metabolism was observed in control transformants.

Autoradiography↗

Investigation of the role of sterol delta 8-->7-isomerase in the sensitivity of Saccharomyces cerevisiae to fenpropimorph.

Treatment of Saccharomyces cerevisiae with the morpholine fungicide fenpropimorph was examined using both a wild-type and a mutant strain (erg2) defective in sterol delta 8-->7-isomerase. No resistance to fenpropimorph was observed in the mutant strain after 3 days, although after 7 days the mutant and the wild-type strains had grown in concentrations of fenpropimorph close to the saturating dose. Re-inoculation of both strains into fresh medium containing fenpropimorph resulted in continued growth and this adaptation to fungicide tolerance was lost on subculture in the absence of fenpropimorph. Analysis of the sterols present in the cells indicated that fenpropimorph treatment resulted in the accumulation of delta 8,14-sterols. This accumulation and the corresponding depletion of ergosterol were correlated with growth inhibition rather than the presence of delta 8-sterols. Together with an absence of gene dosage effect for ERG2 on fenpropimorph sensitivity, this supports the hypothesis that sterol delta 8-->7-isomerase inhibition does not contribute to the fungicidal activity of fenpropimorph.

Base Sequence↗

Studies on azole-induced cell death in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae strain XL16-5B exhibited a fungicidal response to treatment with ketoconazole. Cell death became apparent during prolonged treatment over 72 h following an initial period over 24 h where viable cells were found and limited cell division occurred. Sterol analysis showed some differences between XL16-5B and the strain XY729-5a, which had a fungistatic response to ketoconazole. In particular, the level of ergosterol was higher in XL16-5B and remained high during treatment.

Cytochrome P-450 Enzyme System↗

Release of monokines by pulmonary macrophages following antigen challenge in sensitized guinea pigs.

Guinea pigs were passively sensitized with immune serum to ovalbumin (OA), control serum, or saline. Twenty-four hours later, they inhaled aerosols of OA (2% in saline), saline, or lipopolysaccharide (LPS). Following anesthesia, bronchoalveolar lavage (BAL) was performed at 30, 60, 90 and 120 min postinhalation. Alveolar macrophages (AM) were isolated from the BAL fluid and incubated (18 h) in medium alone or with zymosan (1 mg/ml). Supernatants were collected and levels of interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha) determined by bioassays. Unstimulated AM from animals that inhaled OA, saline, or LPS secreted similar amounts of IL-1 at 30, 60, and 90 min postinhalation. Zymosan (1 mg/ml) significantly increased IL-1 secretion by AM collected at 60 and 90 min from OA-sensitized animals that inhaled OA or saline. AM from guinea pigs sensitized to OA that inhaled OA or LPS secreted significantly increased amounts of IL-6 at 30, 60, 90, and 120 min postchallenge compared to saline sensitized controls. In all groups, AM from LPS-treated animals secreted large amounts of TNF-alpha at all sampling times postchallenge; AM from OA-sensitized and challenged animals secreted increasing amounts of TNF-alpha with time postchallenge, spontaneously and in response to zymosan. By contrast, AM from saline sensitized and challenged guinea pigs did not release detectable amounts of TNF-alpha spontaneously and secreted very low amounts in the presence of zymosan. These findings show that antigen challenge results in a rapid activation of AM isolated from BAL and suggest AM may initiate the development of inflammatory processes associated with antigen challenge.

Administration, Inhalation↗

Benzo(a)pyrene hydroxylase activity in yeast is mediated by P450 other than sterol 14 alpha-demethylase.

Benzo(a)pyrene hydroxylation has been observed in Saccharomyces cerevisiae and the role of sterol 14 alpha-demethylase (CYP51A1) in this activity has been examined by using a strain which contains a gene disruption of CYP51A1. This strain still contained P450 protein(s) with a Soret absorption maximum at 448nm in reduced carbon monoxide difference spectra of the microsomal fraction. On addition of benzo(a)pyrene to this microsomal extract a typical Type I substrate-binding spectrum was obtained and was also observed for the isogenic sister strain containing no CYP51A1 gene disruption. Microsomal extracts of both strains had equivalent activity in the aryl hydrocarbon hydroxylase assay. These results indicate a yeast benzo(a)pyrene hydroxylase activity distinct from sterol 14 alpha-demethylase P450.

Aryl Hydrocarbon Hydroxylases↗

Uveoscleral permeability to intracamerally infused ferritin in eyes of rabbits and monkeys.

The permeability of the uveoscleral outflow pathway from the anterior ocular chamber was examined in rabbit and monkey eyes using anionic ferritin as a tracer. Ferritin, infused intracamerally, had ready access to the choroidal interstitium, and the degree of penetration was generally correlated with the time and pressure relationships during infusion. In both species, there were accumulations of tracer in intercellular spaces at the lamina fusca, but tracer was also present in the sclera. Thus, in contrast to the situation in the eyes of hamsters, the uveoscleral outflow pathway in the eyes of rabbits and monkeys includes the choroidal connective tissue and allows passage of relatively large molecular weight substances.

Animals↗

The expression of human cytochrome P450IA1 in the yeast Saccharomyces cerevisiae.

Data from animal studies suggest that cytochrome P450IA1 catalyses the metabolic activation of several procarcinogenic compounds. In the present study, we have expressed human cytochrome P450IA1 in yeast cells. A 1.70 kb BclI/BamHI fragment containing a full-length human cytochrome P450IA1 cDNA was inserted into the BglII expression site of the yeast expression plasmid pMA91 thereby allowing the ATG initiation codon to be located adjacent to the PGK (phosphoglycerate kinase) promoter. The resulting recombinant plasmid, pCK-1, was introduced into Saccharomyces cerevisiae strains ATCC 44773 and AH22. Microsomes prepared from yeast transformatants of strain ATCC 44773 contained undetectable levels of cytochrome P450. In contrast, microsomes from strain AH22 contained cytochrome P450 with a specific content of 33.3 +/- 10.8 pmol/mg of microsomal protein and showed a reduced carbon monoxide difference spectrum with a peak at 448 nm. Control yeast cells transformed with pMA91 showed no cytochrome P450. Western blots were carried out using an antibody that reacts against rat cytochrome P450IA1 and an antibody that reacts against a synthetic peptide representing a short sequence of human cytochrome P450IA1. A band with a molecular weight of 54 kD was observed in microsomes of yeast transformed with pCK-1, but not with pMA91. When microsomes from yeast transformed with pCK-1 were incubated with benzo(a)pyrene (10 min, 10-160 microM), an estimated Km value of 7 microM was obtained. The availability of yeast cells with functionally active human cytochrome P450IA1 will facilitate molecular structure-activity studies of procarcinogen and drug metabolism by this enzyme in man.

Cell Line, Transformed↗

Structural analysis of potential barriers to bulk-flow exchanges between uvea and sclera in eyes of Macaque monkeys.

The uveal tract of the eyes of monkeys was examined by electron microscopy using both thin sections and freeze-fracture replicas. The ultrastructural features of the lamina fusca in the monkey resembled those previously described for rabbit. The lamina fusca was composed of numerous interleaved processes of fibroblastic and pigmented cells and contained tight junctions between fibroblastic cell processes that were predominantly discontinuous, as well as numerous fenestrations through the attenuated cell processes. There was no regional compaction of cellular processes traversing the entire uvea at the level of the ora serrata as reported previously in hamster eyes.

Animals↗

Structural analysis of potential barriers to bulk-flow exchanges between uvea and sclera in eyes of rabbits.

The uveal tract of rabbit eyes was examined using transmission electron microscopy of thin sections and freeze-fracture replicas. Emphasis was placed on the organization of pigmented and fibroblastic cells near the ora serrata and more generally in the lamina fusca, since these regions were previously found to restrict ingress of tracer molecules to the choroid in hamster eyes. There were significant differences between rabbit and hamster in the disposition of the pigmented cells and fibroblastic layers, the extent and patterns of tight junctions adjoining fibroblastic cells, and the presence of vesicles or fenestrations in the attenuated fibroblastic cell processes.

Animals↗

The effect of gold treatment on monocyte interleukin-1 production in rheumatoid arthritis. A prospective study.

Monocyte interleukin-1 (IL-1) production in vitro was studied in 49 patients with rheumatoid arthritis (RA) and 31 controls. Twenty-six of the RA patients were studied prospectively for up to 12 months after beginning chrysotherapy. About half of the patients (group 1) exhibited pretreatment levels of monocyte IL-1 secretion (as measured by bioassay or B-IL-1) significantly higher than that of the controls. Immunoreactive IL-1 (IR-IL-1) levels, however, were similar to controls. Clinical improvement in this group of patients was modest and transient but could be associated with a fall in the level of IL-1 (B-IL-1 and IR-IL-1) secretion. Other RA patients (group 2) appeared to have normal or reduced pretreatment levels of IL-1 secretion. Chrysotherapy resulted in significant clinical improvement within 3 months, and this was associated with an increase in IL-1 (both B-IL-1 and IR-IL-1) secretion by the patients' blood monocytes to normal or supranormal levels. Thus these two groups of RA patients (which differed only in the average duration of disease) had different prognoses in relation to chrysotherapy and the effect of chrysotherapy-induced remission on monocyte IL-1 secretion was opposite. These results suggest that monocyte IL-1 production in vitro reflects changes secondary to the anti-rheumatic effects of chrysotherapy.

Adult↗

Comparative controlled trial of low-dose weekly methotrexate versus azathioprine in rheumatoid arthritis: 3-year prospective study.

Fifty-three patients with rheumatoid arthritis who required immunosuppressive therapy were commenced in a randomized trial comparing azathioprine to weekly oral pulse methotrexate. After an initial 24-week period, both groups had significantly improved from baseline measures of pain and functional capacity and there were no significant differences in clinical outcomes between the two groups. Laboratory variables of disease activity showed a significant improvement in haemoglobin and ESR in the methotrexate group. Subsequently, the patients were followed for up to 3 years. After one year, more than half of the patients in both groups had discontinued therapy due to inefficacy or adverse events. Adverse effects were more frequent in the patients treated with methotrexate, but withdrawal rates were similar in both groups. From these data, the probability of a patient continuing therapy with either agent for greater than 18 months is low.

Activities of Daily Living↗