Inhibition of mitochondrial respiration by flavoskyrin, a toxic metabolite of Penicillium islandicum Sopp

105

~o~~co~o~ LefEers, 30 (1986) 105-l 11 Eisevier

TOXLett. 1532

INHIBITION OF MITOCHONDRIAL RESPIRATION BY FLAVOSKYRIN, A TOXIC METABOLITE OF PENICILLIUM ISLANDICUh4 SOPP. (Unscheduled

DNA synthesis; genotoxi~ity; uncoupler;

mitochondria)

KIYOSHI KAWAF’*, YOSHINORI NOZAWAa, HIDEKI MORlb and YUKIO OGIHARAC Departments of aBiochemistry and bPathology, Gifu University School of Medicine, Gifu 500, and ‘Department of Pharmacognosy, Faculty of Pharmaceutical Science, Nagoya City University, Nagoya (Japan) (Received August 26th, 1985) (Revision received November 25th. 1985) (Accepted December 4th, 1985)

SUMMARY The effects of flavosk~in, a toxic bianthraquinoid compound from Pen~~il~~urn island~eum Sopp., on the DNA repair system in rat and mouse hepatocytes and on the ATP biosynthesis system in rat hver mitochondria were studied to gain insight into the mechanism for its cytotoxicity. Ftavoskyrin did not elicit unscheduled DNA synthesis (UDS) in hepatocytes at all, implying non-genotoxicity of this compound. Flavoskyrin was found to uncouple oxidative phosphorylation in mitochondria, significantly decreasing both respiratory control (RC) index and P/O ratio. In addition to its uncoupling effect, flavoskyrin produced a marked depression of state-3 respiration, showing 50% inhibition at about 20 pM. These reactions lead to the inhibition of ATP biosynthesis in mitochondria, which may reflect one of the mechanisms of flavoskyrin cytotoxicity.

INTRODUCTION

P. islandicum Sopp. produces a number of quinone and quinoid compounds as secondary metabolites ([ I,21 and references cited therein). One of the metabolites, (- ) luteoskyrin and ( - ) rugulosin were well documented to be toxic and carcinogenic fl,2]. As a molecular mechanism for the cytotoxic action of (-) luteoskyrin, its obstructive effect on ATP biosynthesis in mitochondria has been of*To whom correspondence

should be addressed.

Abbreviations: BSA, bovine serum albumin; DMFA, N,N-dimethylformamide; HPC, hepatocyte primary culture; RC, respiratory control; SMP, submitochondrial particles; TdR, thymine deoxyribonucleotide; UDS, unscheduled DNA synthesis. 037%4274/86/$ 03.50 0 Elsevier Science Publishers B.V. (Biomedical Division)

106

OH

0

C--JFLAVOSKYRI Fig.

1. Structure

OH

N

of flavoskyrin.

fered [3]. Emodin and skyrin, which are also metabolites of the fungus, are cytotoxic to murine leukemia L-1210 culture cells and inhibit ATP synthesis in isolated rat liver mitochondria by exerting an uncoupling effect on oxidative phosphorylation 141. Flavoskyrin (Fig. l), a bianthraquinoid pigment from P. isfandicum Sopp. [5,6] is cytotoxic to HeLa cells, L cells, and isolated rat liver cells, showing 50% inhibition at 20-40 PM [7]. In the present study, the effect of flavoskyrin on oxidative phosphorylation in rat liver mitochondria was examined to assess the mechanism of its cytotoxicity. In addition to the inhibitory effect on the ATP synthesis system in mitochondria, the genotoxic activity of flavoskyrin was studied using rat and mouse HPC cells. Additionally to mutagenicity test [8] which employs the Salmonella/ microsome system, a genotoxicity test has recently been developed as a reliable short-term, in vitro test system using rat and mouse hepatocytes to detect chemical carcinogens [9-l 11. Genotoxic carcinogens have been demonstrated by the test system to significantly enhance the UDS in nuclei due to the activation of DNA repair system, showing a clear correlation between the genotoxicity and carcinogenicity of chemicals (9-111. Carcinogenic fungal products such as aflatoxin Br, sterigmatocystin, and related compounds have also been shown to be positive in this test system [12,13]. MATERIALS

AND METHODS

Reagents Flavoskyrin from P. islandicum Sopp. [6], a gift from Prof. S. Shibata of Meiji College of Pharmacy, was dissolved in DMFA. ADP, Tris, and BSA were products of Sigma Chemical Co. [3H]TdR was obtained from Amersham, U.K. Other reagents from Nakarai Chemical Co. were of the analytical grade.

HPC/DNA

repair test

The test was performed

according

to the procedure

of Williams

[lo]. Hepatocytes

107

were isolated from livers of adult ACI/N rats weighing 200-250 g and of adult C3H/HeN mice weighing about 20 g. The isolated hepatocytes were allowed to attach for 2 h to plastic coverslips in Williams’ medium E (Flow Laboratories, Australia). The cultures were then washed and exposed to the test compounds and [methyl-3H]TdR (10 @/ml, 49 Ci/mmol) for 20 h. All compounds were dissolved in DMFA. N-2-Fluorenylacetamide and diethylnitrosamine were used for rat and mouse liver cells, respectively, as the positive chemicals. At the end of incubation, the cultures were washed and the coverslips mounted on glass slides. The slides were dipped in Sakura NR-M2 photographic emulsion and were exposed for I4 days. Autoradiographic grains were counted in an Olympus counter (type S) with a microscopic attachment. The data were expressed as the average net counts for 3 coverslips +- the standard deviation (50 cells/coverslip~. The test compounds were considered positive when the mean net nuclear grain count was statistically greater than that of the controls and above 5 grains per nucleus. Preparation of rat liver mitochondria and submitochondrial particles and measurement of their respiration Rat liver mitochondria were prepared by the method of Schneider [ 141 using 0.25 M sucrose solution which contained 0.5 mM EDTA and 10 mM Tris-HCl (pH 7.4). SMP were prepared from rat liver mitochondria according to the procedure of Ruzicka and Crane [15]. The respiration of mitochondria and SMP were measured using Galvani-type oxygen electrode (Sensanics Japan Co.) with a reaction mixture composed of 675 ymol sucrose, 30 pmol KCl, 1.5 pmol inorganic phosphate, 1.5 ,umol EDTA, 60 pmol Tris-HCl, and 1.2 mg mitochondrial or SMP protein in a final volume of 3.1 ml (pH 7.4). The reaction was initiated by adding 15 pmol succinate and was carried out at 30°C. Additions were performed as depicted in Fig. 2. State-3 respiration (ADP-driven respiration), state-4 respiration (respiration without added ADP), RC index (ratio of state-3 to state-4 respiration), and P/O ratio (ratio of added ADP to the amount of oxygen consumed during state-3 respiration) were calculated from oxygraph according to Chance and Williams [16]. Protein was determined by the method of Lowry et al. [17] using BSA as a standard protein. RESULTS AND DISCUSSION

The effect of flavoskyrin on UDS in hepatocytes Flavoskyrin was examined for genotoxicity, measured as UDS in hepatocyte primary culture (Table I). Control experiments with N-2-fluorenylacetamide and diethylnitrosamine exhibited UDS in hepatocytes from rat and mouse livers, respectively, indicating their potent genotoxicity. Flavoskyrin did not elicit UDS, indicating its non-genotoxicity, as was the case with emodin and skyrin [12].

108

TABLE

1

RESULTS

OF DNA REPAIR

Compound

TEST WITH

RAT AND MOUSE

Dose (PM)

Flavoskyrin

240 24 2.4

Diethylnitrosaminea

N-2-Fluorenylacetamide”

HEPATOCYTES

Mouse

Rat

UDS grains/nucleus

(% of UDS positive

-0.7 -0.6

f 1.3 (0) + 1.2 (0)

-0.9 -0.8

* +

-1.1

* 1.4 (0)

-1.2

t

100

21.6 + 7.7 (98)

10

11.6 + 4.0 (94)

Positive Mean

-1.1

control controls

for the test in amouse

1.4 (0)

cell)

59.0 + 11.1 (100)

100

28.1 + * -1.0

10 Solvent

1.0 (0) 1.3 (0)

+- 1.2 (0)

7.8

(99)

1.1

(0)

and %at.

+ S.D. from 2 experiments.

Effect of flavoskyrin on oxidative phosphorylation in mitochondria The effect of flavoskyrin on oxidative phosphorylation in isolated rat liver mitochondria was studied using an oxygen electrode (Fig. 2). Freshly prepared mitochondria exhibited a tightly coupled respiration displaying a high RC index and P/O ratio (curve 1). The addition of flavoskyrin caused significant decreases in both RC index and P/O ratio, accompanying a marked depression of state-3 respiration (curve 2). When flavoskyrin at a very high concentration was added, the ADPdriven respiration (state-3 respiration) was no longer observed, showing neither

Succinate

15pmol

AD? 880nmol

I

3 min

4

Fig. 2. Effect of flavoskyrin ditions

are described

on mitochondrial

in detail in MATERIALS

respiration

oxidizing

AND METHODS.

succinate

as substrate.

Reaction

con-

109

respiratory control nor phosphorylation activity (curve 3). The RC index and P/O ratio of mitochondrial respiration were measured in the presence of flavoskyrin at various concentrations (Fig. 3). RC index and P/O ratio were progressively decreased according to the increase of flavoskyrin concentrations, showing a stronger effect on RC index than on P/O ratio. The effect of flavoskyrin on mitochondrial respiration was also examined at concentrations higher than those shown in Fig. 3, but the RC index and P/O ratio could not be accurately calculated due to the severe depression of state-3 respiration at such high concentrations (Fig. 4). The effects of flavoskyrin on succinate oxidases in intact mitochondria and in SMP were compared (Fig. 4). The succinate oxidase in intact mitochondria (state-3 respiration) was strongly inhibited by flavoskyrin, causing 50% inhibition at about 20 PM (curve l), whereas the succinate oxidase in SMP, which are reverted membrane vesicles of mitochondrial inner membranes, was not obviously affected (curve 2), indicating no direct interaction of flavoskyrin with the respiratory chain in mitochondria. The mitochondrial respiration oxidizing L-glutamate, which is a substrate for NAD-linked respiratory chain, was also strongly depressed and NADH oxidase in SMP was again merely slightly affected by flavoskyrin (not shown) as observed for succinate oxidases. The inhibition of flavoskyrin on mitochondrial respiration was no longer reversed by 2,4-dinitrophenol, a typical uncoupling agent [18] which is able to release the mitochondrial respiration depressed by an energytransfer inhibitor such as oligomycin [19] (not shown), indicating non-interference

RC

index

J 1.0

0

10 FLAVOSKVRIN

30

20 (JIM

Fig. 3. The effect of flavoskyrin ditions

50

100

FLAVOSKVRIN

on RC index and P/O ratio of mitochondrial on succinate

were the same as in Fig. 2. Inhibition

ment without in SMP.

0

1

150 0Jt.i

respiration.

200 )

Reaction

con-

and in SMP. Reaction

con-

were the same as in Fig. 2.

Fig. 4. The effect of flavoskyrin ditions

40

flavoskyrin.

Curve 1: succinate

oxidases

in intact mitochondria

rate was calculated oxidase

from the corresponding

in intact mitochondria;

control

Curve 2: succinate

experioxidase

110

with the energy-transfer system in mitochondria. These results indicate that flavoskyrin inhibits mitochondrial respiration by mechanism(s) other than the interferences with the electron-transport and energy-transfer systems, as observed for several uncoupling agents [20-221. The inhibition mechanism, was however, not investigated in further detail because of limited amount of sample supplied. The inhibition by flavoskyrin of mitochondrial ATP synthesis at the concentrations similar to those for the cytotoxic activity may be one of the mechanisms of the drug’s cytotoxicity. ACKNOWLEDGEMENT

The authors are grateful to Prof. S. Shibata of Meiji College of Pharmacy his gift of flavoskyrin.

for

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