Panagrellus Redivivus, Turbatrix Acet1 and Caenorhabditis Elegans
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Comp. Biochem. Physiol. Vol. 86B, No. 1, pp. 103-107, 1987 0305-0491/87 $3.00+0.00 Printed in Great Britain Pergamon Journals Ltd STEROL METABOLISM IN THE NEMATODES PANAGRELLUS REDIVIVUS, TURBATRIX ACET1 AND CAENORHABDITIS ELEGANS DAVID J. CHITWOOD, WILLIAM R. LUSBY and THOMAS A. SALT* Insect and Nematode Hormone Laboratory, Agricultural Research Service, USDA, Building 467, BARC-E, Beltsville, MD 20705, USA and *Department of Botany, University of Maryland, College Park, MD 20742, USA (Received 24 March 1986) Abstract--1. Panagrellus redivivus, Turbatrix aceti and Caenorhabditis elegans were sterilely propagated in semidefined media containing sitosterol or cholesterol, and sterols were isolated and identified by capillary gas-liquid chromatography-mass spectrometry. 2. Each species was capable of removal of the C-24 ethyl substituent of sitosterol and production of 4x -methylsterols. 3. Other modifications of the sterol nucleus varied among the species, as only T. aceti and C. elegans introduced AT-and A~14)-bonds significantly. 4. P. redivivus and, to a much lesser extent, 7". aceti reduced AS-bonds to produce substantial quantities of cholestanol and 4~-methylcholestanol. INTRODUCTION positions of plant-parasitic nematodes with their hosts has indicated that these parasites are probably Unlike higher plants and vertebrates, nematodes are capable of phytosterol dealkylation and nuclear incapable of synthesizing sterols de novo and con- saturation (Chitwood et al., 1985; Cole and Krus- sequently possess a nutritional requirement for sterol berg, 1967; Orcutt et al., 1978). In addition, the (Bolla et al., 1972; Cole and Krusberg, 1968; Comley free-living nematode Turbatrix aceti converts radio- and Jaffe, 1981; Dutky et aL, 1967; Hieb and labelled sitosterol (24~-ethylcholest-5-en-3fl-ol) to Rothstein, 1968). Many structurally diverse sterols radiolabelled 7-dehydrocholesterol (Cole and Krus- satisfy this requirement, including several plant berg, 1968). The purpose of the present investigation sterols (Bolla et al., 1972; Chitwood et al., 1986; was to further characterize sterol metabolism in Dutky et al., 1967; Hieb and Rothstein, 1968). Plant this organism and another free-living nematode, sterols, or phytosterols, unlike most common animal Panagrellus redivivus, by capillary gas chromatog- sterols, generally contain alkyl (i.e. methyl, methyl- raphy-mass spectrometry (GC-MS). Additionally, ene, ethyl, ethylidene) substituents at C-24 of their because Lu et al. (1977) reported maximum re- side chains (Nes and McKean, 1977). production of T. aceti to occur at a lower sterol Interest in nematode sterol biochemistry has been concentration than that used in our previous experi- stimulated by the discovery that several compounds ments with C. elegans, we have replicated some of our with potent nematicidal activity towards various work with C. elegans at a lower concentration of plant- and animal-parasitic and free-living nematodes dietary sterol (10/~g/ml vs 25#g/ml) to facilitate (Bottjer et al., 1984, 1985; Douvres et al., 1980; intergeneric comparison. Feldmesser et al., 1976; Lozano et al., 1984) disrupt sterol metabolic pathways in Caenorhabditis elegans (Chitwood et al., 1984; Lozano et al., 1984, 1985b). This free-living nematode removes the C-24 MATERIALS AND METHODS alkyl substituents of many structurally diverse phytosterols, the major metabolites are usually Dietary sterols 7-dehydrocholesterol (cholesta-5,7-dien-3/~-ol), chol- Sitosterol contained no apparent impurities as determined esterol (cholest-5-en-3fl-ol) and lathosterol (cholest- by thin-layer chromatography (TLC) but contained 1.5% 7-en-3fl-ol) (Chitwood et al., 1984; Lozano et al., campesterol (24~x-methylcholest-5-en-3~-ol) by gas-liquid 1985a). In certain cases, however, other sterols com- chromatography (GLC). No impurities were detected in prise major proportions of the total sterol pool dietary cholesterol by either TLC or GLC. The dietary (Lozano et al., 1985a). In addition, C. elegans cholesterol was supplemented with [4-t4C]cbolesterol, which attaches a single methyl group to the nucleus of the had been obtained from Amersham Corp. (Arlington Heights, IL, USA) and purified via column chromatography sterol molecule to produce significant quantities of to exceed 99% purity by GLC and TLC, and was used at 4~t-methylcholest-8(14)-en-3fl-ol (Chitwood et al., a specific activity of 0.40 Ci/mol. 1983). This direct nuclear methylation pathway has not been reported to occur in any other organism. Nematode culture Sterol metabolism in other nematode species is less P. redivivus, T. aceti and C. elegans were propagated well understood. Comparison of the sterol corn- axenically at 22°C in an aqueous medium containing 103 104 DAVID J. CmrwooD et al. 30mg/ml soy peptone, 30mg/ml yeast extract, 10mg/ml Table 1. Sterol content of Panagrellus redivivus, Turbatrix aceti and dextrose, 0.5 mg/ml hemoglobin, 0.5 #l/ml Tween 80 and Caenorhabditis elegans propagated in media containing 10#g/ml 10/t g/ml sterol, with yeast extract, dextrose and hemoglobin sitosterol or cholesterol,,expressed as percentage of nematode dry extracted previously to remove endogenous sterols (Chit- weight wood et al., 1984). In addition, the medium for T. aceti contained 70 mM acetic acid. Living nematodes from late supplemented sterol logarithmic phase cultures were isolated by centrifugation, sltosterol cholesterol flotation on 40% sucrose solution, and repeated rinsing with distilled water. P. redivivus 0.16 0.12 T. aceti 0.12 0.13 Sterol analysis ~. ele~ans 0.I0 * Lipids were extracted from duplicate samples of 200-400 mg lyophilized nematodes by homogenization in a Ten-Broeck tissue grinder 3 times with chloroform/ *Experiment not performed. methanol (2:1) and subsequent partition against 0.85% NaCI (Folch et al., 1957). Lipid extracts were separated on RESULTS columns of silica gel 60 (70-230 mesh, E. Merck, Darm- stadt, FRG) into neutral lipids (eluted with chloroform) and Gravimetric quantification indicated that the polar lipids (eluted with methanol). The neutral lipids were nematode lipid extracts comprised 12.1% to 15.6%, saponified as described previously (Lozano et al., 1984) and 16.0% to 20.6% and 15.8% to 17.8% of the dry fractionated on 5.0g columns (I.0cm i.d.) of Florisil weight of P. redivivus, T. aceti, and C. elegans, (60-100 mesh, J. T. Baker, Philipsburg, NJ, USA) deacti- vated with 70 H20. Compounds were eluted with 50ml respectively. Sterol content, measured by GLC, var- hexane, 50 ml 1% diethyl ether in hexane (v/v), 50 ml and ied from 0.10% to 0.16% of nematode dry weight 100 ml portions of 5% ether, 50 ml of 20% ether, and 50 ml (Table 1). of ether. The 4g-methylsterols were in the second 5% ether All sterols isolated from nematodes were identical fraction and 4-desmethylsterols were in the 200 ether to authentic reference compounds (except for five fraction. All column chromatographic separations were subsequently described compounds for which we monitored by TLC on Anasil H plates (Analabs, North lacked reference material) by GLC relative retention Haven, CT, USA) developed with hexane/ether/acetic acid times (RRTs, Table 2) of both free sterols and steryl (80:20: 1). Sterols were quantified and tentatively identified acetate derivatives, argentation TLC, and GC-MS. by GLC and analyzed further by acetylation overnight in pyridine/acetic anhydride (3:1) at room temperature. The steryl acetates were purified on Florisil columns similar to Table 2. Gas-liquid chromatographic relative retention times of those described previously and were eluted with 5% diethyl sterols from Panagrellus redivivus, Turbatrix aceti or Caenorhabditis ether in hexane. The acetates were analyzed further by elegans, expressed relative to cholesterol. GLC was performed isothermally on a DB-I fused silica capillary column argentation column chromatography (Chitwood et al., (14m x 0.32 mm i.d., 0.25,um film) and on a packed glass column 1985), argentation TLC (Chitwood et al., 1984), GLC, UV (2m x 2mm i.d.) containing 2.0% OV-17 stationary phase spectroscopy, and GC-MS. Sterol DB-I 0V-17 Instrumentation GLC of sterols and steryl acetates was performed iso- Cholesta-5,7,g(lll-trienol 0.98 1.06 thermally with a Shimadzu model GC-9A gas chro- Cholest-8(14)-enol o.g9 1.02 matograph fitted with a 14m x 0.32 mm i.d. J & W DB-1 Cholesterol 1.00 1.00 fused silica capillary column (0.25/am film) and connected to a Shimadzu model C-R3A recording integrator, as well Cholestanol 1.02 1.02 as a Varian model 3700 instrument equipped with a packed Cholest-8(9)-enol 1.05 1.05 glass column (2m × 2mm i.d.) containing 2.0% OV-17 Desmosterol 1.08 1.20 liquid phase and connected to a Shimadzu C-R 1B recording integrator. Flame ionization detectors were used for quan- 7-Dehydrocholesterol 1.10 1.16 titative analyses; for radioactivity determinations, we Lathosterol 1.12 1.17 trapped the effluent from a Varian 3700 gas chromatograph Cholesta-8(g),24-dienol 1.15 1.27 fitted with a thermal conductivity detector and a 9.8 m x 0.75 mm i.d. Supelco SPB-1 glass capillary column Cholesta-5,7,24-trienol 1.21 1.40 (1.0 #m film). Compounds from the GLC effluent (as well Cholesta-7,24-dienol 1.23 1.42 as all column chromatographic fractions) were radio- analyzed in a xylene-based scintillation fluid (ScintiLene, Campesterol 1.30 1.32 Fisher Scientific, Fairlawn, NJ, USA) and counted in a Fucosterol 1.60 1.72 Beckman LS 5801 liquid scintillation system, with quench in Sitosterol 1.60 1.63 each sample measured with the H-number technique. Cap- illary GC-MS of steryl acetates with a DB-1 column and Stigmastanol 1.63 1.65 UV spectroscopy were performed as described previously 4a-Methyl cholest-8(14)-enol 1.18 1.15 (Lozano et aL, 1984).