Natural Anthracenone Subcellular Distribution and Effects on Nadph-Cytochrome P450 Reductase Microsomal Activity

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<ul><li><p>DRUG AND CHEMICAL TOXICOLOGY, 19(4), 301-312 (1996) </p><p>BRIEF COMMUNICATION </p><p>NATURAL ANTHRACENONE SUBCELLULAR DISTRIBUTION </p><p>AND EFFECTS ON NADPH-CYTOCHROME P450 REDUCTASE </p><p>MICROSOMAL ACTIVITY </p><p>Martha Guerrero-Olazarin*, Jose M. Viader-Salvadb* </p><p>Departamento de Farmacologia y Toxicologia, Facultad de Medicina, U.A.N.L. </p><p>Monterrey, N. L. (Mexico) </p><p>ABSTRACT </p><p>Natural anthracenone subcellular distribution and effects on NADPH- </p><p>cytochrome P450 reductase microsomal activity. Subcellular distribution study of </p><p>a natural anthracenone (T-5 14) isolated from Kurwinskiu humboldtiunu showed </p><p>to be homogeneous on subcellular (nuclear, mitochondrial, peroxisomal and </p><p>microsomal) fractions prepared from rat liver treated with an acute dose of T-5 14. </p><p>These results indicate that T-5 14 can pass easily through subcellular compartment </p><p>membranes and an absence of selectivity for some subcellular organelles. A </p><p>significant increase of protein on liver homogenates and NADPH-cytochrome </p><p>P450 reductase microsomal activity indicates that T-5 14 may act as a microsomal </p><p>*Present address: Departamento de Bioquimica, Facultad de Medicina, U.A.N.L., Monterrey, N.L. (M6xico). </p><p>301 </p><p>Copyright 0 1997 by Marcel Dekker, Inc. </p><p>Dru</p><p>g an</p><p>d C</p><p>hem</p><p>ical</p><p> Tox</p><p>icol</p><p>ogy </p><p>Dow</p><p>nloa</p><p>ded </p><p>from</p><p> info</p><p>rmah</p><p>ealth</p><p>care</p><p>.com</p><p> by </p><p>Frei</p><p>e U</p><p>nive</p><p>rsita</p><p>et B</p><p>erlin</p><p> on </p><p>10/3</p><p>1/14</p><p>For </p><p>pers</p><p>onal</p><p> use</p><p> onl</p><p>y.</p></li><li><p>302 GUERRERO-OLAZARAN AND VIADER-SALVADO </p><p>enzymatic inducer. In addition, this enzymatic specific activity increment could be </p><p>due to the interaction of T-5 14 with the microsomal redox cycling. </p><p>INTRODUCTION </p><p>Karwinskia humboldtiana (buckthorn or tullidora) is a shrub of the </p><p>Rhamnaceae family distributed over Mexican territory, southwestern United </p><p>States and in Central America1-2. The ingestion of the fruit causes a progressive </p><p>symmetric and ascendent paralysis, in both animals and man, similar to Guillain- </p><p>B a d Syndrome and in some cases ending with death3-7. Intoxication with this </p><p>plant has been traditionally considered as a regional human and livestock </p><p>epidemiological problem and it is calculated that in the states of Tamaulipas and </p><p>Nuevo Le6n, Mexico, approximately 6-8 intoxication cases with paralysis occur </p><p>annually, almost exclusively in children7. Dreyer et a1.8 isolated and characterized </p><p>four toxic principles from the fruits of the plant, which have been typified as </p><p>anthracenones being designated as T-544, T-496, T-5 14 an T-5 16 according to </p><p>their molecular weight. Previous studies have reported the presence of severe </p><p>hepatopulmonar lesions in different animal ~pecies79~ with the whole fruit, as </p><p>well as with toxins T-514 and T-544. T-514 was found to be toxic for liver, lung </p><p>and kidney, without showing any manifestations of neurological damage. A </p><p>mortality rate of 100% was observed in CD1 mice with 1.5-2.5 g k g oral doses </p><p>of green fruit containing 0.7-0.8% T-5 14 at 48 hr and with 12 mgkg oral doses </p><p>of purified toxin in less than 16 hr7t10. In the present report, the distribution of </p><p>T-5 14 in subcellular organelles isolated from rat liver treated with acute dose of </p><p>T-5 14 was evaluated in order to determine its affinity to a specific organelle. The </p><p>specific activity of the microsomal enzyme NADPH-cytochrome P-450 reductase </p><p>Dru</p><p>g an</p><p>d C</p><p>hem</p><p>ical</p><p> Tox</p><p>icol</p><p>ogy </p><p>Dow</p><p>nloa</p><p>ded </p><p>from</p><p> info</p><p>rmah</p><p>ealth</p><p>care</p><p>.com</p><p> by </p><p>Frei</p><p>e U</p><p>nive</p><p>rsita</p><p>et B</p><p>erlin</p><p> on </p><p>10/3</p><p>1/14</p><p>For </p><p>pers</p><p>onal</p><p> use</p><p> onl</p><p>y.</p></li><li><p>NATURAL ANTHRACENONE SUBCELLULAR DISTRIBUTION 303 </p><p>(E.C. 1.6.2.4) was determined in order to elucidate the participation of </p><p>microsomal enzymes in the metabolism and biological mechanism of T-5 14. </p><p>MATERIALS AND METHODS </p><p>T-5 14 was isolated from Kanvinskia humboldtiuna in the Pharmacology </p><p>and Toxicology Department of the School of Medicine, Autonomous University </p><p>of Nuevo Le6n following the method of Guerrero et al.ll. Adult female Wistar </p><p>rats weighing 200 f 19 g were treated, after 12 hr fast with a single </p><p>intraperitoneally dose (20 mg/kg body weight, 114% of LD5,)12913 of T-5 14 in </p><p>ethanol (5 mg/ml). Ethanol was administered to the control animals. All animals </p><p>were sacrificed by cervical dislocation 4 hr after administration. Livers were </p><p>removed, weighed and rinsed until free of blood with homogenization buffer </p><p>(0.25 M sucrose, 50 mM Tris-HC1 and 1 mM EDTA pH 7.4). The livers were </p><p>minced and homogenized in homogenization buffer (4 ml/g tissue) using a Potter- </p><p>Elvehjem-type homogenizer. The homogenate was strained through four layers </p><p>of cheesecloth and then the filtrate was centrifuged (CRU-5000 centrifuge IEC) </p><p>for 15 min at 800 g. Nuclear, mitochondrial, peroxisomal and microsomal </p><p>fractions were isolated from the supernatant obtained as described by Singh </p><p>Poulosl4 using a fixed angle Ti-70 rotor and a L5-75B Beckman ultracentrifuge </p><p>(Beckman Instruments, San Ramon, California, U.S.A.). The isolated fractions </p><p>were suspended in 5 ml of homogenization buffer and stored at -20C until </p><p>analysis. </p><p>Homogenate and each subcellular fraction were sonificated for 10 sec and </p><p>T-5 14 was extracted using C18 Sep-Pack cartridge (Waters Associates, Milford, </p><p>Dru</p><p>g an</p><p>d C</p><p>hem</p><p>ical</p><p> Tox</p><p>icol</p><p>ogy </p><p>Dow</p><p>nloa</p><p>ded </p><p>from</p><p> info</p><p>rmah</p><p>ealth</p><p>care</p><p>.com</p><p> by </p><p>Frei</p><p>e U</p><p>nive</p><p>rsita</p><p>et B</p><p>erlin</p><p> on </p><p>10/3</p><p>1/14</p><p>For </p><p>pers</p><p>onal</p><p> use</p><p> onl</p><p>y.</p></li><li><p>304 GUERRERO-OLAZARAN AND VIADER-SALVAD~ </p><p>Massachusetts, U.S.A.). To precondition the cartridge, 3 rnl of methanol and 3 </p><p>ml of deionized water were added. One to 3 ml of sample were added to the </p><p>preconditioned cartridge, washed by passing 6 mi of deionized water and then air </p><p>pushed through to dry the sorbent bed. All the fractions collected to this point </p><p>were discarded. T-514 was eluted by passing 3 ml of chloroform through the </p><p>cartridge. The organic eluate was evaporated to dryness under nitrogen stream </p><p>and reconstituted in 75 p1 of benzene for subsequent quantification of T-514. </p><p>Quantification of T-5 14 extracted from subcellular fraction was made by </p><p>thin-layer chromatography using reflectance densitometry according to the </p><p>method previously described in the literaturellvl5 with a Carl Zeiss </p><p>chromatogram spectrophotometer model QM I11 attached to an Auto-lab </p><p>minigrator. The protein concentrations were evaluated by the Lowry method16 </p><p>using bovine serum as standard. </p><p>NADPH-cytochrome P450 reductase activity was determined by monitoring </p><p>changes in absorbance at 550 nm in a Acta I11 dual beam spectrophotometer </p><p>(Beckman Instruments, San Ramon, California, U.S.A.) following the reduction </p><p>of cytochrome c as described by Lakel7. </p><p>All chemicals were purchased from Sigma Chemical Co. (St. Louis, </p><p>Missouri, U.S.A.) while the analytical grade solvents and the TLC aluminum </p><p>sheets (silica gel 60 F254) were purchased from Merck (Darmstadt, Germany). </p><p>RESULTS AND DISCUSSION </p><p>The preparations of subcellular organelles from rat liver treated with T-5 14 </p><p>and controls were characterized by morphological observations under electron </p><p>Dru</p><p>g an</p><p>d C</p><p>hem</p><p>ical</p><p> Tox</p><p>icol</p><p>ogy </p><p>Dow</p><p>nloa</p><p>ded </p><p>from</p><p> info</p><p>rmah</p><p>ealth</p><p>care</p><p>.com</p><p> by </p><p>Frei</p><p>e U</p><p>nive</p><p>rsita</p><p>et B</p><p>erlin</p><p> on </p><p>10/3</p><p>1/14</p><p>For </p><p>pers</p><p>onal</p><p> use</p><p> onl</p><p>y.</p></li><li><p>ANTHRACENONE SUBCELLULAR DISTRIBUTION 305 </p><p>CD NU MI PX MC CT Subcellular fractions </p><p>FIGURE 1 </p><p>T-5 14 subcellular fraction distribution evaluated as % with respect to the liver homogenate. Values are the mean f SEM (n=6). CD: Cellular debris NU: Nuclear fraction MI: Mitochondrial fraction PX: Peroxisomal fraction MC: Microsomal fraction CT: Cytosol </p><p>microscopy. This characterization shows that the cellular debris (sediment </p><p>obtained at 800 g) was formed mainly by complete cells, erythrocytes, damaged </p><p>cells and some nucleii; nuclear fraction (sediment obtained at 2,500 g) was </p><p>constituted mainly by nucleii and some damaged cells; mitochondria1 fraction </p><p>(sediment obtained at 9,600 g) was formed by pure and some damaged </p><p>mitochondria; peroxisomal fraction (sediment obtained at 16,500 g) was formed </p><p>by light mitochondria, lisosomes and peroxisomes; microsomal fraction </p><p>(sediment obtained at 105,000 g) was constituted by microsomes from the rough </p><p>endoplasmic reticulum as well as smooth endoplasmic reticulum; cytosol </p><p>Dru</p><p>g an</p><p>d C</p><p>hem</p><p>ical</p><p> Tox</p><p>icol</p><p>ogy </p><p>Dow</p><p>nloa</p><p>ded </p><p>from</p><p> info</p><p>rmah</p><p>ealth</p><p>care</p><p>.com</p><p> by </p><p>Frei</p><p>e U</p><p>nive</p><p>rsita</p><p>et B</p><p>erlin</p><p> on </p><p>10/3</p><p>1/14</p><p>For </p><p>pers</p><p>onal</p><p> use</p><p> onl</p><p>y.</p></li><li><p>306 GUERRERO-OLAZARAN AND WADER-SALVADO </p><p>loo 1 </p><p>HC HT CDC CDT NUC NUT MIC MIT PXC PXT MCC MCT CTC CTT </p><p>Subcellular fractions </p><p>FIGURE 2 </p><p>Amount of protein per gram of tissue of subcellular fractions for controls and treated animals. Values are the mean &amp; SEM (n=6). H: Liver homogenate PX: Peroxisomal fraction CD: Cellular debris CT: Cytosol MI: Mitochondrial fraction - C: Control MC: Microsomal fraction - T: Treated NU: Nuclear fraction </p><p>(supernatant obtained at 105,000 g) was constituted by the cytoplasmic soluble </p><p>fraction. </p><p>Figure 1 shows T-5 14 subcellular fraction distribution. The nuclear and </p><p>microsomal fraction has a slightly higher concentration of T-5 14 than the other </p><p>subcellular fractions. These results indicate an absence of selectivity for some </p><p>subcellular organelles. The homogeneous distribution of the substance in the </p><p>studied subcellular fractions is in accordance with the results obtained from </p><p>pharmacokinetic studiesl5 that showed a high distribution throughout the body. </p><p>Dru</p><p>g an</p><p>d C</p><p>hem</p><p>ical</p><p> Tox</p><p>icol</p><p>ogy </p><p>Dow</p><p>nloa</p><p>ded </p><p>from</p><p> info</p><p>rmah</p><p>ealth</p><p>care</p><p>.com</p><p> by </p><p>Frei</p><p>e U</p><p>nive</p><p>rsita</p><p>et B</p><p>erlin</p><p> on </p><p>10/3</p><p>1/14</p><p>For </p><p>pers</p><p>onal</p><p> use</p><p> onl</p><p>y.</p></li><li><p>NATURAL ANTHRACENONE SUBCELLULAR DISTRIBUTION 307 </p><p>16 l8 1 </p><p>T </p><p>T </p><p>T </p><p>HC HT MCC MCT Subcellular fractions </p><p>FIGURE 3 </p><p>NADPH-cytochrome P450 reductase specific activity of controls and treated animals. Values are the mean HC: Control liver homogenate MCC: Control microsomal fraction HT: Treated liver homogenate MCT: Treated microsomal fraction </p><p>SEM (n=6). </p><p>Besides, these results point out that T-514 can pass easily through subcellular </p><p>compartment membranes. The amount of protein per gram of tissue of subcellular </p><p>fractions for controls and treated animals are presented in figure 2 and were not </p><p>significantly different. However, the liver homogenates of treated animals </p><p>showed a significant increase of protein quantity (p &lt; 0.1). This protein elevation </p><p>in the liver homogenate can be explained as T-5 14 acting as an enzymatic inducer </p><p>and the small increase being due to a short exposition period. </p><p>The results of NADPH-cytochrome P-450 reductase specific activity </p><p>obtained are resumed in figure 3. The specific activity of this enzyme in the </p><p>treated animals was greater (1.6 times) than the control preparations (p &lt; 0.05). </p><p>Dru</p><p>g an</p><p>d C</p><p>hem</p><p>ical</p><p> Tox</p><p>icol</p><p>ogy </p><p>Dow</p><p>nloa</p><p>ded </p><p>from</p><p> info</p><p>rmah</p><p>ealth</p><p>care</p><p>.com</p><p> by </p><p>Frei</p><p>e U</p><p>nive</p><p>rsita</p><p>et B</p><p>erlin</p><p> on </p><p>10/3</p><p>1/14</p><p>For </p><p>pers</p><p>onal</p><p> use</p><p> onl</p><p>y.</p></li><li><p>308 GUERRERO-OLAZARAN AND VIADER-SALVADO </p><p>OH OH 0 </p><p>FIGURE 4 </p><p>T-5 14 chemical structure </p><p>If we take into account the structural characteristics of T-514 as a polycydic </p><p>aromatic compound (figure 4) and that T-514 is a lipid-soluble substance at </p><p>physiological pH (fat/water partition coefficient 8.26, solubility in water and </p><p>chloroform of 0.01 mg/l and 20 mg/ml, respectively)lg, this molecule could be a </p><p>potential microsomal enzymatic system inducer. In addition, structural studies </p><p>carried out on T-5 I4 intoxicated animals for longer exposition periods have </p><p>shown proliferation of the endoplasmic reticulum, cytoplasmatic fat deposits in </p><p>hepatocytes'g, high hepatic accumulation of T-5 14 (unpublished results), the </p><p>protein increase in the liver homogenate and the specific activity increase of </p><p>NADPH-cytochrome P-450 reductase indicate that T-5 14 belongs to the class of </p><p>Dru</p><p>g an</p><p>d C</p><p>hem</p><p>ical</p><p> Tox</p><p>icol</p><p>ogy </p><p>Dow</p><p>nloa</p><p>ded </p><p>from</p><p> info</p><p>rmah</p><p>ealth</p><p>care</p><p>.com</p><p> by </p><p>Frei</p><p>e U</p><p>nive</p><p>rsita</p><p>et B</p><p>erlin</p><p> on </p><p>10/3</p><p>1/14</p><p>For </p><p>pers</p><p>onal</p><p> use</p><p> onl</p><p>y.</p></li><li><p>NATURAL ANTHRACENONE SUBCELLULAR DISTRIBUTION 309 </p><p>inducer drugs such as phenobarbital. The specific activity increase of NADPH- </p><p>cytochrome P-450 reductase found in such a short exposure time is a better </p><p>marker to indicate that T-514 acts as a microsomal enzymatic inducer than to </p><p>measure total microsomal protein increase. In addition, this increase in enzymatic </p><p>specific activity could be due to the interaction of T-514 with the microsomal </p><p>redox cycling and the possible formation of T-5 16semiquinone radical and </p><p>reactive oxygen metabolites (02- , OH, H202) that might be implicated in the </p><p>toxicity mechanism of T-5 14*'. </p><p>ACKNOWLEDGMENTS </p><p>We thank Dr. med. Alfred0 Piiieyro-L6pez, Chairman of the Department of </p><p>Pharmacology and Toxicology, School of Medicine, Autonomous University of </p><p>Nuevo Le6n for his support of our research, T.L.C. Laura M. Escobar-Gonzilez </p><p>and Q.F.B. Teresa Zanatta-Calder6n for their technical assistance, Prof. R.M. </p><p>Chandler-Burns for his critical reading of our manuscript and Mrs. Patricia </p><p>Hernindez-Sierra for her help in typing the MS. </p><p>WFERENCES </p><p>R. Fernhdez-Nava, Tres especies nuevas de Kurwinskiu (Rhamnaceae) de </p><p>MCxico, Acta bothica Mexicana, 2, 1 1 (1 988) </p><p>R. Fernindez Nava, Nombres comunes, usos y distribuci6n Geogrifica del </p><p>GCnero Kurwinskiu (Rhamnaceae) en Mexico, Anales Inst. biol. Univ. </p><p>Nac. Aut6n. Mexico, 63 1, 1 (1992). </p><p>Dru</p><p>g an</p><p>d C</p><p>hem</p><p>ical</p><p> Tox</p><p>icol</p><p>ogy </p><p>Dow</p><p>nloa</p><p>ded </p><p>from</p><p> info</p><p>rmah</p><p>ealth</p><p>care</p><p>.com</p><p> by </p><p>Frei</p><p>e U</p><p>nive</p><p>rsita</p><p>et B</p><p>erlin</p><p> on </p><p>10/3</p><p>1/14</p><p>For </p><p>pers</p><p>onal</p><p> use</p><p> onl</p><p>y.</p></li><li><p>310 GUERRERO-OLAZARAN AND VIADER-SALVADO </p><p>10 </p><p>A. Escobar and D. Niteto, Aspectos Neuropatol6gicos de la intoxicacidn </p><p>con Karwinskia humboldtiana. Estudio experimental. Gaceta MCdica, </p><p>MCxico, 95 2, 163 (1965). </p><p>K. Charlton and K. Pierce, A Neuropathy in Goats Caused by Experimental </p><p>Coyotillo (K. humboldtiuna) Poisoning (111), Pathology Veterinary, 7, 408 </p><p>(1970). </p><p>K. Aoki and E.J. Mufioz-Martinez, Quantitative changes in myelin proteins </p><p>in a peripheral neuropathy caused by tullidora (Kunuinskia humboldtiana), </p><p>J. Neurochem, 36, 1 (1981). </p><p>K. Aoki and E.J. Mufioz-Martinez, On the increase of the 68,000 dalton </p><p>polypeptide in the tullidora (buckthorn) neuropathy, J. Neurobiol, 14,463 </p><p>(1983). </p><p>M.V. Bermudez, D. Gonzilez-Spencer, M. Guerrero, N. Waksman and A. </p><p>Piiieyro, Experimental intoxication with fruit and purified toxins of </p><p>buckthorn (Kanuinskia humboldtiana), Toxicon, 24 109 I (1986). </p><p>D. Dreyer, I. Arai, C. Bachman, W. Anderson, R. Smith and D. Daves, </p><p>Toxins causing non-inflammatory paralytic neuropathy . Isolation and </p><p>structure elucidation, J. Am. Chem. SOC., 97, 4985 (1975). </p><p>M. V. Bermudez, F. J. Martinez, M. E. Salazar, N. Waksman and A. </p><p>Piiieyro, Experimental acute intoxication with ripe fruit of Karwinskia </p><p>humboldtiana (tullidora) in rat guinea pig, hamster and dog, Toxicon,. 3, 1493 (1992). </p><p>N. Waksman, L. Martinez, and R. Fernindez, Screening quimico y </p><p>toxicoldgico de otras especies del gtnero Karwinskia, Rev. Latinoaam. </p><p>Quim., a , 2 7 (1989). </p><p>Dru</p><p>g an</p><p>d C</p><p>hem</p><p>ical</p><p> Tox</p><p>icol</p><p>ogy </p><p>Dow</p><p>nloa</p><p>ded </p><p>from</p><p> info</p><p>rmah</p><p>ealth</p><p>care</p><p>.com</p><p> by </p><p>Frei</p><p>e U</p><p>nive</p><p>rsita</p><p>et B</p><p>erlin</p><p> on </p><p>10/3</p><p>1/14</p><p>For </p><p>pers</p><p>onal</p><p> use</p><p>...</p></li></ul>

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