DRUG AND CHEMICAL TOXICOLOGY, 19(4), 301-312 (1996)

BRIEF COMMUNICATION

NATURAL ANTHRACENONE SUBCELLULAR DISTRIBUTION

AND EFFECTS ON NADPH-CYTOCHROME P450 REDUCTASE

MICROSOMAL ACTIVITY

Martha Guerrero-Olazarin*, Jose M. Viader-Salvadb*

Departamento de Farmacologia y Toxicologia, Facultad de Medicina, U.A.N.L.

Monterrey, N. L. (Mexico)

ABSTRACT

Natural anthracenone subcellular distribution and effects on NADPH-

cytochrome P450 reductase microsomal activity. Subcellular distribution study of

a natural anthracenone (T-5 14) isolated from Kurwinskiu humboldtiunu showed

to be homogeneous on subcellular (nuclear, mitochondrial, peroxisomal and

microsomal) fractions prepared from rat liver treated with an acute dose of T-5 14.

These results indicate that T-5 14 can pass easily through subcellular compartment

membranes and an absence of selectivity for some subcellular organelles. A

significant increase of protein on liver homogenates and NADPH-cytochrome

P450 reductase microsomal activity indicates that T-5 14 may act as a microsomal

*Present address: Departamento de Bioquimica, Facultad de Medicina, U.A.N.L., Monterrey, N.L. (M6xico).

301

Copyright 0 1997 by Marcel Dekker, Inc.

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302 GUERRERO-OLAZARAN AND VIADER-SALVADO

enzymatic inducer. In addition, this enzymatic specific activity increment could be

due to the interaction of T-5 14 with the microsomal redox cycling.

INTRODUCTION

Karwinskia humboldtiana (buckthorn or tullidora) is a shrub of the

Rhamnaceae family distributed over Mexican territory, southwestern United

States and in Central America1-2. The ingestion of the fruit causes a progressive

symmetric and ascendent paralysis, in both animals and man, similar to Guillain-

B a d Syndrome and in some cases ending with death3-7. Intoxication with this

plant has been traditionally considered as a regional human and livestock

epidemiological problem and it is calculated that in the states of Tamaulipas and

Nuevo Le6n, Mexico, approximately 6-8 intoxication cases with paralysis occur

annually, almost exclusively in children7. Dreyer et a1.8 isolated and characterized

four toxic principles from the fruits of the plant, which have been typified as

anthracenones being designated as T-544, T-496, T-5 14 an T-5 16 according to

their molecular weight. Previous studies have reported the presence of severe

hepatopulmonar lesions in different animal ~pecies79~ with the whole fruit, as

well as with toxins T-514 and T-544. T-514 was found to be toxic for liver, lung

and kidney, without showing any manifestations of neurological damage. A

mortality rate of 100% was observed in CD1 mice with 1.5-2.5 g k g oral doses

of green fruit containing 0.7-0.8% T-5 14 at 48 hr and with 12 mgkg oral doses

of purified toxin in less than 16 hr7t10. In the present report, the distribution of

T-5 14 in subcellular organelles isolated from rat liver treated with acute dose of

T-5 14 was evaluated in order to determine its affinity to a specific organelle. The

specific activity of the microsomal enzyme NADPH-cytochrome P-450 reductase

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NATURAL ANTHRACENONE SUBCELLULAR DISTRIBUTION 303

(E.C. 1.6.2.4) was determined in order to elucidate the participation of

microsomal enzymes in the metabolism and biological mechanism of T-5 14.

MATERIALS AND METHODS

T-5 14 was isolated from Kanvinskia humboldtiuna in the Pharmacology

and Toxicology Department of the School of Medicine, Autonomous University

of Nuevo Le6n following the method of Guerrero et al.ll. Adult female Wistar

rats weighing 200 f 19 g were treated, after 12 hr fast with a single

intraperitoneally dose (20 mg/kg body weight, 114% of LD5,)12913 of T-5 14 in

ethanol (5 mg/ml). Ethanol was administered to the control animals. All animals

were sacrificed by cervical dislocation 4 hr after administration. Livers were

removed, weighed and rinsed until free of blood with homogenization buffer

(0.25 M sucrose, 50 mM Tris-HC1 and 1 mM EDTA pH 7.4). The livers were

minced and homogenized in homogenization buffer (4 ml/g tissue) using a Potter-

Elvehjem-type homogenizer. The homogenate was strained through four layers

of cheesecloth and then the filtrate was centrifuged (CRU-5000 centrifuge IEC)

for 15 min at 800 g. Nuclear, mitochondrial, peroxisomal and microsomal

fractions were isolated from the supernatant obtained as described by Singh

Poulosl4 using a fixed angle Ti-70 rotor and a L5-75B Beckman ultracentrifuge

(Beckman Instruments, San Ramon, California, U.S.A.). The isolated fractions

were suspended in 5 ml of homogenization buffer and stored at -20°C until

analysis.

Homogenate and each subcellular fraction were sonificated for 10 sec and

T-5 14 was extracted using C18 Sep-Pack cartridge (Waters Associates, Milford,

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304 GUERRERO-OLAZARAN AND VIADER-SALVAD~

Massachusetts, U.S.A.). To precondition the cartridge, 3 rnl of methanol and 3

ml of deionized water were added. One to 3 ml of sample were added to the

preconditioned cartridge, washed by passing 6 mi of deionized water and then air

pushed through to dry the sorbent bed. All the fractions collected to this point

were discarded. T-514 was eluted by passing 3 ml of chloroform through the

cartridge. The organic eluate was evaporated to dryness under nitrogen stream

and reconstituted in 75 p1 of benzene for subsequent quantification of T-514.

Quantification of T-5 14 extracted from subcellular fraction was made by

thin-layer chromatography using reflectance densitometry according to the

method previously described in the literaturellvl5 with a Carl Zeiss

chromatogram spectrophotometer model QM I11 attached to an Auto-lab

minigrator. The protein concentrations were evaluated by the Lowry method16

using bovine serum as standard.

NADPH-cytochrome P450 reductase activity was determined by monitoring

changes in absorbance at 550 nm in a Acta I11 dual beam spectrophotometer

(Beckman Instruments, San Ramon, California, U.S.A.) following the reduction

of cytochrome c as described by Lakel7.

All chemicals were purchased from Sigma Chemical Co. (St. Louis,

Missouri, U.S.A.) while the analytical grade solvents and the TLC aluminum

sheets (silica gel 60 F254) were purchased from Merck (Darmstadt, Germany).

RESULTS AND DISCUSSION

The preparations of subcellular organelles from rat liver treated with T-5 14

and controls were characterized by morphological observations under electron

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ANTHRACENONE SUBCELLULAR DISTRIBUTION 305

CD NU MI PX MC CT Subcellular fractions

FIGURE 1

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

microscopy. This characterization shows that the cellular debris (sediment

obtained at 800 g) was formed mainly by complete cells, erythrocytes, damaged

cells and some nucleii; nuclear fraction (sediment obtained at 2,500 g) was

constituted mainly by nucleii and some damaged cells; mitochondria1 fraction

(sediment obtained at 9,600 g) was formed by pure and some damaged

mitochondria; peroxisomal fraction (sediment obtained at 16,500 g) was formed

by light mitochondria, lisosomes and peroxisomes; microsomal fraction

(sediment obtained at 105,000 g) was constituted by microsomes from the rough

endoplasmic reticulum as well as smooth endoplasmic reticulum; cytosol

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306 GUERRERO-OLAZARAN AND WADER-SALVADO

loo 1

HC HT CDC CDT NUC NUT MIC MIT PXC PXT MCC MCT CTC CTT

Subcellular fractions

FIGURE 2

Amount of protein per gram of tissue of subcellular fractions for controls and treated animals. Values are the mean & 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

(supernatant obtained at 105,000 g) was constituted by the cytoplasmic soluble

fraction.

Figure 1 shows T-5 14 subcellular fraction distribution. The nuclear and

microsomal fraction has a slightly higher concentration of T-5 14 than the other

subcellular fractions. These results indicate an absence of selectivity for some

subcellular organelles. The homogeneous distribution of the substance in the

studied subcellular fractions is in accordance with the results obtained from

pharmacokinetic studiesl5 that showed a high distribution throughout the body.

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NATURAL ANTHRACENONE SUBCELLULAR DISTRIBUTION 307

16 l8 1

T

T

T

HC HT MCC MCT Subcellular fractions

FIGURE 3

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

SEM (n=6).

Besides, these results point out that T-514 can pass easily through subcellular

compartment membranes. The amount of protein per gram of tissue of subcellular

fractions for controls and treated animals are presented in figure 2 and were not

significantly different. However, the liver homogenates of treated animals

showed a significant increase of protein quantity (p < 0.1). This protein elevation

in the liver homogenate can be explained as T-5 14 acting as an enzymatic inducer

and the small increase being due to a short exposition period.

The results of NADPH-cytochrome P-450 reductase specific activity

obtained are resumed in figure 3. The specific activity of this enzyme in the

treated animals was greater (1.6 times) than the control preparations (p < 0.05).

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308 GUERRERO-OLAZARAN AND VIADER-SALVADO

OH OH 0

FIGURE 4

T-5 14 chemical structure

If we take into account the structural characteristics of T-514 as a polycydic

aromatic compound (figure 4) and that T-514 is a lipid-soluble substance at

physiological pH (fat/water partition coefficient 8.26, solubility in water and

chloroform of 0.01 mg/l and 20 mg/ml, respectively)lg, this molecule could be a

potential microsomal enzymatic system inducer. In addition, structural studies

carried out on T-5 I4 intoxicated animals for longer exposition periods have

shown proliferation of the endoplasmic reticulum, cytoplasmatic fat deposits in

hepatocytes'g, high hepatic accumulation of T-5 14 (unpublished results), the

protein increase in the liver homogenate and the specific activity increase of

NADPH-cytochrome P-450 reductase indicate that T-5 14 belongs to the class of

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NATURAL ANTHRACENONE SUBCELLULAR DISTRIBUTION 309

inducer drugs such as phenobarbital. The specific activity increase of NADPH-

cytochrome P-450 reductase found in such a short exposure time is a better

marker to indicate that T-514 acts as a microsomal enzymatic inducer than to

measure total microsomal protein increase. In addition, this increase in enzymatic

specific activity could be due to the interaction of T-514 with the microsomal

redox cycling and the possible formation of T-5 16semiquinone radical and

reactive oxygen metabolites (02- , OH, H202) that might be implicated in the

toxicity mechanism of T-5 14*'.

ACKNOWLEDGMENTS

We thank Dr. med. Alfred0 Piiieyro-L6pez, Chairman of the Department of

Pharmacology and Toxicology, School of Medicine, Autonomous University of

Nuevo Le6n for his support of our research, T.L.C. Laura M. Escobar-Gonzilez

and Q.F.B. Teresa Zanatta-Calder6n for their technical assistance, Prof. R.M.

Chandler-Burns for his critical reading of our manuscript and Mrs. Patricia

Hernindez-Sierra for her help in typing the MS.

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NATURAL ANTHRACENONE SUBCELLULAR DISTRIBUTION 311

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