326

Biochimica et Biophysica Acta, 1092 (1991) 326-335 © 1991 Elsevier Science Publishers B.V. 0167-4889/91/$03.50 ADONIS 0167488991001611

BBAMCR 12922

Effect of metal ion catalyzed oxidation of rifamycin SV on cell viability and metabolic performance of isolated rat hepatocytes G u i l l e r m o T. Sfiez ~, Victoria Vails ~, H u g o C a b e d o ~, A n t o n i o Iradi 2, William H. Bannister 2 a n d Joe V. Bannister 3,4 I

R Department of Biochemistry and Molecular Biology, Faculty of Medicine. University of Valencia. Valencia (Spare). ' Department of Physiology, Faculty of Medicine, University of Valencia, Valencia (Spare). " Department of Biomedical Sciences, UplwersiO' of Maim. M~ida (Malta) and ~ Biotechnologv Centre, Cranfield Institute of Technology. Cranfield, Bedfordshire (U. K. )

(Received 7 December 1990)

Key words: Rifamycin SV; Metal ion; Oxygen free radical; Glutathione; Cell viability; Carbohydrate metabolism; (Rat hepatocyte)

The effect of rifamycin SV on metabolic performance and cell viability was studied using isolated bepatocytes from fed, starved and glutathione (GSH) depleted rats. The relationships between GSH depletion, nutritional status of the cells, glucose metabolism, lactate dehydrogenase (LDH) leakage and malondialdehyde (MDA) production in the presence of rifamycin SV and transition metal ions was investigated. Glucose metabolism was impaired in isolated hepatocytes from both fed and starved animals, the effect is dependent on the rifamycin SV concentration and is enhanced by copper (II). Oxygen consumption by isolated bepatocytes from starved rats was also increased by copper (lI) and a partial inhibition due to catalase was observed. Cellular GSH levels which decrease with increasing the rifamycin SV concentration were almost depleted in the presence of copper (lI). A correlation between GSH depletion and LDH leakage was observed in fed and starved cells. Catalase induced a slight inhibition of the impairment of gluconeogenesis, GSH depletion and LDH leakage in starved bepatocytes incubated with rifamycin SV, iron (il) and copper (ll) salts. Lipid peroxidation measured as MDA production by isolated bepatocytes was also augmented by rifamycin SV and copper (ll), especially in hepatic cells isolated from starved and GSH depleted rats. Higher cytotoxicity was observed in isolated hepatocytes from fasted animals when compared with fed or GSH depleted animals, it seems likely that in addition to GSH level, there are other factors which may have an influence on the susceptibility of hepatic cells towards xenobiotic induced cytotoxicity.

Introduction Quinone containing substances are a widely ranging family of naturally occurring compounds with anticancer activity [1]. Quinone containing antibiotics can be enzymatically activated to free radical semiquinones that either react directly with biological targets, such as DNA and RNA, or generate cytotoxic oxygen-dependent superoxide and hydroxyl radicals [2,31. Free radical production during drug oxidation has been shown to initiate lipid peroxidation in a variety of experimental

Abbreviations: GSH, reduced glutathione; LDH, lactate dehydrogenase: MDA, malondialdehyde; TBA, thiobarbituric acid. Correspondence: G.T. S~ez, Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Valencia, 46010 Valencia, Spain.

systems [4]. However, notwithstanding that the wide clinical potential of quinone containing antibiotics against a broad spectrum of pathological microorganisms or as antineoplasmic agents has been experimentally defined, their use has been hindered by several forms of tissue damage which may be a result of druginduced free radical formation [5,61. Rifamycin SV is a naphthohydroquinone with antibiotic activity isolated from the microorganism Notocardia mediterranei [7]. The use of rifamycin SV as a particularly efficient agent against Mycobacterium tuberculosis [8] has been known for almost two decades. Recently, this drug has been reported to have other interesting antiviral [9], anti-inflammatory [10l and immunosuppressive [11] properties. Both the hydroquinone moiety and the long aliphatic bridge of rifamycin SV are responsible for its pharmaco-chemical behaviour. The long aliphatic bridge of the antibiotic is responsible for the binding to RNA polymerase [12]

327 which consequently impairs bacterial DNA-dependent RNA synthesis [13,14]. On the other hand, during oxidation, hydroquinone moiety (QH2) yields the semiquinone radical ( Q H - ) which can reduce oxygen to form the superoxide radical. The rate of the reaction is increased in the presence of divalent transition metal ions resulting in the production of hydrogen peroxide and hydroxyl radicals [15,161. Oxidative destruction of DNA by rifamycin SV and copper (II) has been related to the copper-dependent produc~,!' n of hydrogen peroxide as a result of its metal ion catalyzed oxidation [17]. However, in spite of the clinical use of rifamycin SV and the relative amount of information about its pharmacodynamic properties its effect on the metabolic status and integrity of cells has not been investigated. Liver xenobiotic detoxification is due to the presence of GSH-utilising enzymes such as glutathione-S-transferase and glutathione peroxidase. Thiolate anions are effective nucleophiles and can undergo substitution reactions either directly or catalyzed by glutathione-Stransferase [18]. Acting as hydrogen donor, GSH contributes to the reduction of hydrogen peroxide and lipid per~,~ades catalysed by glutathione peroxidase [19,20], thereby acting as an inhibitor and repair mechanism of peroxidation induced damage. GSH, therefore,, ;~ one of the most important defences against oxidati,re stress and lipid peroxidation to which liver cells are exposed during drug detoxification [21]. A potentially dangerous situation therefore arises when the level of GSH is depleted. Low levels of GSH have been reported to increase cell susceptibility to lipid peroxidation [22] and metabolic changes [21,23,24]. Lipid peroxidation is increased in mice acutely intoxicated with paracetamol when the hepatic GSH level is decreased by starvation [251. In the present investigation we report the effect of rifamycin SV and metal ions on glucose metabolism, redox state and ATP concentration as well as the leakage of LDH and MDA production of isolated hepatocytes as an indication of their metabolic performance and cell viability, respectively. The role of GSH on the susceptibility of isolated hepatocytes to rifamycin SV was tested using cell suspensions containing different GSH concentrations, i.e., cells from fed and starved rats and GSH depleted cells. Material and Methods

Animals Male Wistar rats weighing 150-200 g were fed ad libitum with a standard diet (Prasa Vara de Cuart, Valencia, Spain) and kept under appropriate conditions. In some experiments rats were starved for 48 h but they had free access to water before hepatocyte isolation.

Chemicals Substrates, enzymes and coenzymes used for incubations or metabolite assays were of analytical grade purchased from various chemical companies. Rifamycin SV, catalase (thymol free), diethylmaleate and buthionine sulphoximine were obtained from Sigma (Dorset, U.K.). Reduced glutathione was from Merck (Darmstadt, F.R.G.) and pyruvate sodium salt was from Boehringer (Mannheim, F.R.G.). The rest of the substrates, enzymes and coenzymes used for metabolite assays and the buffer preparations were from the above mentioned companies. Deionized distilled water was purchased from Quimica Clinica Aplicada, Amposta, Spain. Isolation and incubation of hepatocytes To avoid circadian rhythm-induced metabolic changes, experiments were started around mid-day. Hepatocytes were isolated from fed or starved rats by a simplified version [26] of the perfusion method of Berry and Friend [27]. Cell viability was tested with trypan blue. Approx. 95% of the isolated hepatocytes were found to exclude the dye. Incubation conditions were as described elsewhere [281. 2 ml of hepatocytes suspension containing approx. 2- 10 6 cells in Krebs-Henseleit saline equilibrated with O J C O 2 (95% : 5%) were incubated in a shaking water bath at 37°C in 25 ml conical flasks sealed with rubber stoppers. Incubation mixtures containing cells and appropriate concentrations of substrates or metabolites, were made up to 4 ml final volume with Krebs-Henseleit buffer solution (pH 7.4). Antibiotic, metal ion and enzyme solutions were freshly prepared just before preparation of the hepatocytes suspensions. Incubations were started by the addition of rifamycin SV and metal ions immediately after pipetting of the cells in respective flasks. Reactions were stopped after 60 min with 0.4 ml 20% perchloric acid. To estimate the cell content, a factor of 3.7 was used to convert dry into wet weight. Induction of GSH depletion GSH depleted hepatocytes were isolated from rats treated with both diethylmaleate (0.1 ml/kg) which is a substrate of glutathione-S-transferase [29], and buthionine sulfoximine (1 g/kg), a specific inhibitor of GSH synthesis [30], 1 h prior to the normal isolation procedure. Cell suspensions showing GSH concentrations below 0.5 lamol/g cells or undetectable values were used for incubations as previously described [24]. Analytical methods At the end of the incubation period metabolic reactions were stopped by the addition of 0.4 ml of 20°Z (v/v) perchloric acid to the incubation mixtures and placed on an ice-containing tray. Acidified incubated samples were centrifuged to precipitate proteins and the supernatants were neutralized with a few drops of

328 stabilization of the cell suspension, rifamycin SV, metal ions and catalase were added at various concentrations.

potassium hydroxide for the enzymatic determination of glucose [30], lactate [32], pyruvate [33], acetoacetate [34], and /]-hydroxybutyrate [35]. Constants of 1.11. 10 -4 and 4.93-10 -2 were used to calculate cytosolic and mitochondrial redox states from the ratios of the substrate pairs lactate-pyruvate and ,8.hydroxibutyrateacetoacetate, respectively, as follows: N A D + / N A D H = K × ( [ o x i d i s e d substrate]/[reduced substrate]) [36]. G S H was determined in samples neutralized with sodium bicarbonate [37]. L D H leakage from isolated rat hepatocytes was measured in incubation mixtures which were not treated with acid. T h e s e were centrifuged at slow speed to spin down the cells and appropriate dilutions of the cell-free supernatants were used for determination of activity [38]. Malondialdehyde production was assayed as TBA reactive products following the methodology described by Stacy and Priestley [39].

Statistical treatment of results All results except oxygen consumption measurements, were analysed by means of Student's t-test. Results

Metabolic and cytotoxic interactions of rifamycin S V and copper (II) on isolated hepatocytes from fed rats Rifamycin SV at low concentrations was found to induce a slight increase in glucose production by isolated rat hepatocytes (Table I). This effect was proportional to the concentration of the antibiotic in the incubation medium and enhanced by the presence of copper (II) ions with maximal effect being observed when the rifamycin SV concentration was 1 mM. At 1 mM concentration the increase was of 19.4% in the absence and of 46.9% in the presence of the metal, as compared with the control (at 60 rain). Lactate and pyruvate concentrations were also affected, although the rifamycin SV concentration-dependent effect was not as evident as with glucose release. Rifamycin SV alone did not significantly modify the pyruvate levels. However, the level of this metabolite decreased to half or even less of its control value in the presence of copper (II). The changes observed on lactate concentra-

Oxygen consumption measurements Oxygen uptake measurements on hepatocytes from starved rats were carried out with a Gilson Oxygraph supplied with a thermostated electrode chamber. All assays were performed at 37°C, the electrode was calibrated with 100% air saturation at 3 7 ° C . Hepatocytes suspended in Krebs-Henseleit buffer (pH 7.4) were equilibrated with O2/CO2 (95%:5%) for a few seconds and oxygen uptake for control cells was recorded. In other experiments, after equilibration and

TABLE I Effect of rifamycin SV in the presence of copperon glucoseproduction, pyruvate, lactate, acetoacetat~;[3-hydroxybatyrateand A TP levels and redox state (NAD +/NADH) of isolated hepatotytesfrom fed rats An average of 80 mg (wet weight) of isolated hepatocytes from fed rats was incubated in duplicate 25 ml conical flasks in the presence of a substrate containing mixture made up to a final volume of 4 ml with Krebs-Henseleitbuffer (pH 7.4). Incubations were for 60 rain at 37°C. 0.05 mM of copper (11)as copper sulphate was added immediatelyafter isolation of cells. The range of rifamycin SV concentrations used was from 0.4 to 1 raM, with the increments indicated in the table. All parameters shown were assayed in neutralized pereh[oric acid extracts, as described in Materials and Methods, Results are means+ S,D. with the number of experimentsin parentheses. Statistical significance: * P < 0.05 compared with the control (60 rain):, p < 0.05 and , , p < 0.005 for the effect of copper (ll) compared with each ~'espectiverifamycin SV concentration. Glucose Pyruvate Lactate NAD ÷/ Acetoacetate ~-OH-butyrate (~mol/g ceils) (t~mol/gcells) (/~mol/gcells) NADH (lumol/gcells) (/~mol/gcells) (cytosol) Initial value Control 60rain Rif0.4mM Rif 0.4 mM +Cu(ll) Rif0.6mM Rif 0.6 raM +Cu(ll) Rif0.8mM Rif 0.8 mM +Cu(ll) Rifl mM Rif l mM +Cu(il)

21.0+ 9.6 (3)

1.7+0.3 (3)

7.0-1-0.8 (3) 2 1 5 7 (3) 2 2 3 3 (5) 2 7 7 2

NAD+/ ATP NADH (pmol/g (mitoch.) cells)

1.6+0.3 (3)

0,10+0.05

(3)

325

1.9+0.5 (3)

6.5+1.4 (3) 5.5+0.9 (4)

0.27+0.12 0.12+0.03" (4)

488 930

1.7+0.3 (3) i.6+0.5 (3)

67.8+21.0 (3) 69.65:13.8 (5)

14.0+0.6 (3) 57.0+7.6 15.0+1.3" (5) 52.0-1-5.1

64.8±25.8 (6) 73,8+15.0 (5)

8.7+!.2"*(5) 65.0+-6.3° (5) 1 2 0 6 16.0+1.4" (5) 48.0=t:6.7 (5) 3 0 0 3

5.7+0.8 5.3+0.4

5.00+1.10"*(4) 23 0.05+0.01" (4) 2 1 5 0

1.5+0.4 (4) 1.7+0.5 (4)

97.2+25,2 (6) 97.8:1:9.0 (5)

7.7+1A**(4) 70.0:t:7.1**(4) 991 16.0+-1.9 (4) 59.0+-5.9 (4) 2~3

5.4+1.1 (5) 3.8+_0.7*(5)

4.80+1.10**(5) 0.10+-0.02 (5)

23 771

1.3+0.4 (4) 1.7+_0.4 (4)

99.0+_12.6"(5) 5.5+0.8 n (6) 57.9=1=6.1 (6) 869 81.0+-12.0 (5) 14.0+_2.5 (5) 51.0+_2.0 (5) 2 4 7 3

5.0+-0.7*(5) 6.0+_0.3 (4)

5.10+0.58°* (5) 0.25+_0.07 (4)

20 487

i.3+-0.3 (3) 1.7+_0.5 (3)

99.6_+33.0 (3)

5.5+-0.6 (5)

5.00+-0.65** (5)

32

0.9+_0.0*(3)

5.3+1.2"* (4) 61.0+-7.2" (4)

783

329 tions were in the opposite direction (Table I). Lactate levels were found to increase where the pyruvate levels decreased. It may, therefore, be assumed that the micromoles of pyruvate which disappeared were present in the form of lactate, possibly due to a shift of the lactate dehydrogenase equilibrium towards the lactate pool. In the mitochondria, rifamycin SV alone tended to slightly decrease the levels of/3-hydroxybutyrate, while in the presence of copper (II) a significant increase was observed. However, there was no effect on the acetoacetate concentration in the absence or presence of copper (II), resulting in a decrease of both cytosolic and mitochondrial redox state of these cells (Table I). ATP concentration was not significantly affected except in the presence of 1 mM rifamycin SV and copper (II), where a 5070 decrease was observed. In the same group of incubations a cell viability study correlating GSH levels with LDH leakage and MDA productions was also carried out. As shown in Table II, a proportional increase in the rifamycin SV concentration in the incubation mixture resulted in a proportional decrease in the GSH levels of isolated hepatocytes. However, even at a high concentration of the antibiotic, GSH levels remain above 3 ~tmol/g cells. This may explain the observed relatively low release of L D H and the lack of TBA reactive material production compared with the respective control values as well as with other experimental situations presenting lower concentrations of GSH. However, when rifamycin SV was incubated in the presence of copper (II) a significant decrease in the level of GSH is observed, with values falling below 0.5 /~mol/g cells. In the same group of experiments, a parallel increase in LDH leakage and ME ~. production by isolated cells is ~Iso nb~erved ( T ~. I1).

Effect of rifamycin S V and metal ions on metabolic performance and cell viability of isolated hepatocytes from starved rats Metabolic and cell viability changes were also observed in hepatocytes isolated from starved rats. Fig. 1 shows the inhibitory effect of increasing rifamycin SV concentrations with and without copper (It) on gluconeogenesis resulting from lactate plus pyruvate. In the presence of copper (If) a significant increase of the inhibition effect is observed. To test the effect of rifamycin SV and the possible contribution of hydrogen peroxide produced during its oxidation, hepatocytes were incubated in the presence of a fixed concentration of the antibiotic (0.4 mM), metal ions (0.1 #M) and catalase (0.06 mg/ml). Metabolic and cell viability parameters as well as oxygen consumption were measured for this purpose. Table Ill shows the effect of rifamycin SV, copper (ll) and iron (ll) and iron ( l i d on gluconeogenesis, redox state and ATP levels of hepatocytes incubated in the presence of 10 mM lactate plus 1 mM pyruvate. The basal rate of glucose synthesis from endogenous substrates was 0.11 + 0.02 # m o l / g cells per rain (six experiments) (result not shown). Gluconeogenesis from lactate plus pyruvate is inhibited by approx. 20% in the presence of 0.4 mM rifamycin SV. This inhibition is not affected by iron (IIl), while in the presence of iron (II) or copper (11) a significantly enhanced effect is observed. In the latter case, catalase pre~ents to some degree the impairment of glucose synthesis although the rate of this metabolic pathway still remains low when compared with the control value. The pyruvate concentration also decreases and the addition of catalase resulls in its recovery to a value close to the control. Initial lactate concentration (160 /tmol/g cells (eight

TABLE il

Copper (!1) catalysed effect of rifamycin on GSH level. LDH and MDA production in isolated rat hepatocytes from fed rats Incubation conditions were as described in Table I. Analytical procedures where performed as described in Materials and Methods. Table values are means+ S.D. with the number o[ experiments in parenthesis. Statistical significance. * P < 0.05 and * * P < 0.005 compared with the control (60 rain); • p < 0.05 and e o p < 0.005 for the effect of copper (11) compared with each respective rifamycin SV concentration. Total LDH leakage from digitonin treated hepatocytes was 300/tmol/min per g cells.

Initial value Control 60 rain Rif0.4 Rif 0.4+Cu(ll) Rif0.6 Rif 0.6+Cu(il) Rif0.8 Rif 0.8+Cu(II) Rif 1.0 Rif 1 + Cu(II)

GSH (lamol/g cells)

LDH (U.l./g cells)

70 of digitonin treated cells

TBA-MDA (/tmol/g cells)

4.80.+_0.32 (3) 4.90+0.28 (3) 4.00+0.38* (3) 0.45 +0.02ee (4) 3.80+0.61" (4) 0.36_+0.03ee (5) 3.70_+0.20"*(4) 0.29_+0.01 ee (5) 3.10-+0.48 * *(4) 0.13 + 0.05 e e (3)

47+ 0.8 (3) 56+11.0 (3) 69+ 7.0 (4) 92+ 5.7 ° e (4) 74+ 6.9* (4) ll0_+ 10.0®e(4) 79_+ 8.7" (4) 160_+ 14.0ee (3) 95 _+ 6.6 * *(4) 190-+ 50.0 • (3)

16 19 23 31 25 37 26 53 32 63

0.22+0.05 0.31 +0.02 0.28+0.05 0.29+0.05 0.30_+0.05 0.32_+0.05 0.30_+0.05 0.35+0.01 0.27+0.01 0.35 -+0.03 e e

(3) (3) (5) (6) (5) (5) (3) (3) (5) (3)

330

14

~2

10 c E

0.8

"6 0.6 E =k

0.4g/:

/

x

02

Control

O,4mM

06rnM

08raM

1.0mM

Fig. !. Effect of rifamycin alone and in the presence of cop'per on the rate of gluconeogenesis in isolated hepatocytes, Isolated hepatocytes

from 48 h starved rats were incubated in the presence of tO mM lactate plus I mM pyruvateand increasingconcentrationsof rifamycin SV (0,1-1 mM) in the absence(black bars) and presence(shaded bars) of 0.05 mM CuSO4,Incubationswerecarried out for 60 min at 37°C in duplicate 25 ml conical flasks. Results are means+S.D, of 3-7 individual experiments. Control value represents the rate of glnconeogenesisin the presence of lactate plus pyruvatealone. Initial glucose concentrationat the beginningof the incubation was 1.80+ 0.60 Fmol/g [7]. Statisticalsignificance: * P < 0.005 for the effectof rifamycin SV. o p < 0.05 and o o p < 0.005 for the effect of copper comparedwith rifamycinSVeffect.

experiments)) decreases to 120 Fmol/g cells during 1 h incubation indicating that 40 Fmol/g are metabolised prior to glucose synthesis. However, the lactate concentration is preserved in the presence of rifamycin SV as well as with the addition of metal ions with no appreciable effect being observed due to catalase. The cytosolic redox state of the hepatocytes tends to diminish in the presence of iron (If) and copper (II) and an inhibitory effect is achieved by catalase. The changes observed indicate that the acetoacetate and fl-hydroxybutyrate concentrations differ depending on the type of incubation mixture tested. The slight effect of rifamycin SV on the acetoacetate concentration is enhanced by the addition of iron (Ill) and iron (II) to a different degree depending on the valence of the iron used. However, copper (II) ions were found to increase the acetoacetate concentration to a significant level which returns to the control level when catalase is added to the incubation medium. Catalase also exerts a protective effect with rifamycin SV alone and with rifamycin SV and iron (lII) or iron (If) (Table Ill). ATP levels were only significantly affected when iron (ll) or copper (II) were included, with rifamycin SV, in the incubation mixture. Catalase prevents the slight decrease in ATP concentration in the presence of iron

(II) but it had no effect in the presence of the copper (II) (Table liD. The viability of isolated hepatoytes from starved rats was more susceptible to impairment by rifamycin SV and copper (II), in terms of LDH leakage to the extracellular medium. Incubaticn of hepatocytes in the presence of 0.4 mM rifamycin SV alone resulted in discrete changes in the intracellular GSH concentration and LDH leakage to the incubation medium (Table IV). Iron salts in ferric or ferrous forms slightly enhanced the decrease of GSH concentration induced by rifamycin SV but the GSH level remained above 2 /~mol/g cells. At this high GSH value, no significant effect on LDH release due to iron salts was observed. However, the effect of rifamycin SV in the presence of copper (II) appears to exhibit a cytotoxic situation. Copper (II) added in the presence of rifamycin SV enhanced GSH depletion to levels approx. 20~ of the control, while LDH leakage rose to 65% of the total enzyme concentration. Catalase induced partial protection on both the GSH decrease and LDH leakage but it failed to achieve enough protection against the induced cytotoxicity (Table IV). Oxygen consumption by starved rat hepatocytes increases in the presence of rifamycin SV alone and the effect is enhanced when copper (II) is present (Table V). The addition of catalase to the incubation mixture results in a decrease of cell respiration in all situations tested. This inhibition of oxygen uptake is due to enzymatic breakdown of hydrogen peroxide formed during rifamycin SV oxidation returning oxygen to the incubation medium. This effect supports the role of hydrogen peroxide as a natural product of rifamycin SV oxidation and is possibly responsible, in part, for both metabolic and cytotoxic effects of the antibiotic.

Comparative study of the effect of rifamycin SV and copper (II) on GSH depletion, LDH leakage and TBA reactive material production by isolated hepatocytes from fed, starved and GSH depleted rats. The importance of the nutritional and GSH status of the cell The increase in cytoxicity induced by rifamycin SV in starved rats is very clear especially if we consider the decrease of the antibiotic threshold concentration to achieve its effects on GSH levels and LDH leakage. In Tables II and IV the observed decrease in GSH is correlated with the LDH leakage in hepatocytes from both fed and starved animals incubated in the presence of increasing concentrations of rifamycin SV alone. Although a good correlation is observed with hepatocytes from fed rather than starved rats, it was in the starved rats where a higher cytotoxic situation was established, that a significant increase in LDH leakage rising to approx, a 60% of total LDH found in digitonin treated cells was observed when 0.4 mM rifamycin SV was incubated with 0.05 mM copper.

331 TABLE II1 Effect of rifamvcin S V in the presence of copper and iron on gluconeogenesis, pyruvate, lactate, aeetoacetate, ~-hydroxybutyrate and A TP levels and redox state (NAD ÷/NA DH) of isolated hepatocytes from starved rats Incubations were perfot'med in duplicate flasks under the time, temperature and pH conditions specified in the preceding tables with the following pertinent modifications: to test the gluconeogenic performance of rat hepatocytes, cell suspensions from 48 h starved animals were incubated in the presence of 10 mM lactate plus 1 mM pyruvate as gluconeogenic precursors except for 60 rain control tests which contained 2 ml of cells and 2 ml of Krebs-Henseleit buffer. The rate of gluconeogenesis without added substrates was 0.11 _+0.02 (6) ~umol/g per mix. Reagent concentrations were: rifamycin SV 0.4 raM, metal salts 0.05 mM and catalase 0.06 mg/ml. Values are mean_+ S.D. for the number of experiments in parentheses. Statistical significance: * P < 0.05 and * * P < 0.005 compared with the control (60 min); • p < 0.05 and o® p < 0.005 for the effect of catalase, zx, initial value expressed as # m o l / g cells representing the amount of glucose present in the hepatocytes at the beginning of the incubation.

Initial value Control 60rain Rif Rif +Cat Rif +Fe(lll) Rif + Fe(lll) +Cat Rif +Fe(II) Rif + Fe(ll) +Cat Rif +Cu(il) Rif + Cu(II) +Cat

Glucose (p, mol/g cells per mix)

Pyruvate (/~mol/g celE

Lactate (#mol/g cells)

1.80+0.60 ~ (7)

15.0-+4.3

(9)

160.0+14.0

(9)

1.15+0.11 (6) 0.80-+0.11"* (6)

14.0+-3.0 12.0+1.2

(9) (8)

120.0+-24.0 (9) 140.0+ l l , 0 * (8)

0.87+0.07

(5)

14.0+3.7

(6)

130.0+14.0

0.77+0.09**(3)

14.0+-4.3

(3)

0.72_+0.09

(3)

15.0+3.6

0.60_+0.12"* (5)

0.77_0.08 °

(4)

0.27+0.06 ** (6)

0.52_+0.15® (3)

Acetoacetate fl-OH-butyrate (ttmol/g cells) (/~mol/g cells)

NAD ÷ NADH (mitoch.)

ATP (~mol/g cells)

844

7.6+1.5

(5)

0.78+0.26

(5)

198

2.0+0.3

(10)

1051 772

9.8+2.7 8.5 + 3.0

(6) (7)

2.60+2.10 2.70_+0.59

(6) (7)

76 64

2.1_+0.2 2.1 +0.2

(10) (10)

(6)

970

9.1+2.1

(5)

1.60_+0.56 ° (5)

115

2.1_+0.0

(4)

130.0+-17.0

(3)

970

2.3+-I.1" (3)

1.20+-0.81

(3)

39

2.0_+0.1

(6)

(3)

130.0+15.0

(3)

1039

3.8+0.4

(3)

3.20+1.70

(3)

24

2.0+-0.1

(4)

!1.0+1.3

(3)

150.0+28.0

(3)

660

7.3+1.1

(4)

1.80+0.43

(~)

82

1.8+0.1"

(5)

14.0+5.2

(4)

140.0+25.0

(4)

900

9.9+2.8°e(3)

1.20_+.0.34

(3)

167

2.1+0.2 °

(5)

9.5_+3.6 * (7)

140.0+21.0

(7)

611

12.0+3.5

(7)

3.50+0.21

(7)

69

1.5+0.2"* (6)

140.0+16.0

(4)

772

8.8+1.0

(4)

1.80+0.16°®(4)

99

1.0+0.4 °

12.0+1.5

(4)

NAD+/ NADH (cytosol)

Fig. 2 summarises the threhsold concentration effect o f r i f a m y c i n SV for t h e c y t o t o x i c i n t e r a c t i o n in t h e t h r e e cell p o p u l a t i o n s p r e s e n t i n g d i f f e r e n t i n i t i a l G S H

(4)

c o n t e n t s . G S H level a n d v i a b i l i t y o f i s o l a t e d h e p a t o cytes was affected by increasing rifamycin SV concentration. This effect was dramatically enhanced by

TABLE IV Role of catalase on metal ion dependent GSH release and LDH leakage induced by rifamycin S V in isolated hepatocytes from starved rats Cell viability of isolated rat hepatocytes from starved rats was investigated by the quantification of LDH leakage in cell-free supernatants obtained by slow centrifugation of the samples and compared with the levels of GSH found in whole cell extracts. Reagent concentration were: rifamycin SV 0.4 raM, metal salts 0.05 mM and catalase 0.06 mg/ml. Results are means+S.D, showing the number of experiments in parentheses. Statistical significance: * P < 0.05 and * * P < 0.005 for the effect of rifamycin SV and metal ions compared with the control (60 rain): e p < 0.05 and oe p < 0,005 for the effect of catalase. Total LDH leakage from digitonin treated hepatocytes was 500/~mol/min per g cells.

Initial value Control 60 min Rif Rif+Cat Rif+Fe(III) Rif+Fe(lII)+Cat Rif+Fe(ll) Rif+Fe(II)+Cat Rif + Cu(ll) Rif+Cu(ll)+Cat

GSH ( p,mol/g cells)

% of control

3.3+0.77 (12) 3.1 +0.61 (11) 2.7+0.40 (9) 3.1+0.73 (4) 2.1+0.46" (3) 2.7+0.98 (3) 2.1+0.44" (4) 2.8+0.48 (4) 0.8 + 0.l 3 * * (5) 1.2+0.17 * (3)

100 87 100 68 87 68 90 24 39

LDH (# tool/rain per g cells)

7o of digitonin treated cells

48+_ 4.5 (6) 50+ 4.5 (6) 59+ 1.7" (6) 46+ 4.3**(5) 56+ 4.8 (4) 46+ 4.3* (3) 68+ 7.4* 53+ 5.6* (4) 330 + 72.0 * *(3) 260+-43.0 (3)

10 10 12 9 ll 9 14 11 66 52

332 GSH DEPLETED (C)

STARVED (B)

FED (A)

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Fig, 2. Concentration-coupe effect o[ rifamycin SV on cell viability and its relation to the nutritional situation and GSH levels of isolated rat hepatccytes and the role o[ copper (II) on rifamycin SV induced cell damage. Isolated hepatocytes from fed, starved and GSH depleted rats were incubated during 60 rain in the presence o[ increasing concentrations of rifamycin gV (0.4-1.0 raM) alone (black bars), and in the presence of rifamycin SV plus 0,05 mM Cu$O4 (shaded bars), with the incubation conditions reported in Materials and Methods. Results are means + S.D. (vertical bars) of 3-12 experiments. Statistical significance: * P < 0.05 and * * P < 0.005 comparing values obtained in starved and GSH depleted cells with the corresponding values of the fed cells, Total I.DH leakage from digitonin treated hepatocytes corresponds to an activity of 500 /Jmol/min per g cells (0 and 60 rain represent values at the beginning and at the end of the incubation without added substrates, respectively).

the presence of a fixed concentration of copper (II) (0.05 pM). The leakage of LDH also increases (especially in the presence of the metal) exhibiting a parallel with the decrease of GSH in fed and starved rats. Malondialdehyde production was less affected in the hepatocytes isolated from fed rats as compared with starved or GgH depleted rats. Initial GSH values were lower in the hepatocytes obtained from starved rats than in fed rats and at similar rifamycin SV concentrations the depletion of GSH was greater in starved cells (Fig. 2A and B). In this case, lower levels of GSH were accompanied by higher values of LDH activity in the respective cell-free supernatants. Maximal LDH leakage was observed when GSH levels fell below 1.0 /~mol/g cells. However, in GgH depleted hepatocytes, with almost undetectable values of glutathione (Fig. 2C), cell viability was more compromised than in the fed cells but less if compared

with the starved cells. Fig. 2B shows how the threshold response to rifamycin SV concent,'ation is greater in the hepatocytes isolated from starved rats. A maximal increase of LDH leakage which corresponds to the total amount of enzyme concentration obtained from digitonin treated cells (corresponding to an activity of 550 #mol/min per g cells) was observed using the highest rifamycin SV concentration (1 mM) in the presence of 0.05 mM copper. These values were significantly higher than in the fed rats although GSH levels could also be depleted below 1 # m o l / g cells providing that copper (II) was also present in the incubation mixture containing fed cells. In the presence of 0.4 mM rifamycin plus 0.05 copper (II), GSH is depleted in the fed and starved hepatocytes to 0.4 and 0.8 # m o l / g cells, respectively. However, this small difference in the GSH values does not correlate with the observed LDH leakage, i.e., corresponding to

333 TABLE V

Oxygen consumption of isolated rat hepatocytes in presence of rifamycin S V. Effect of metal ions and catalase Oxygen uptake measured by Clark type electrode at 37 ° C (pH 7.4). Final volume of the incubation mixture was 1.8 ml containing isolated hepatoeytes from starved rats and added reagents at the following final concentrations: rifamycin SV 0.4 mM; CuSO4, FeCIa.6H20 and (NH4)2FeSO 4 0.05 raM; eatalast 0.06 mg/ml. Measurements were performed for at least 15 rain. Values are representative results of more than three experiments not differing from each other by more than 5%. Oxygen uptake (it tool 0 2/rnin per g cells) Control Rif Rif + Cat R i f + Fe(lll) Rif+ Fe(lll)+ cat Rif + Fe(ll) Rif+ Fe(ll) + cat Ri f + Cu(l I) Rif+Cu(ll)+cat

I.C' + 0.06 1.52 + 0.09 1.39 + 0.09 1.27+0.01 1.19+0.02 1.34 + 0.11 1.08+0.13 1.69 + 0.04 1.55 +0.03

an activity of 92 #mol/min per g for cells from fed against 330 #mol/min per g for cells from starved rats (Table II and IV). This difference is observed throughout the range of rifamycin SV concentrations (Fig. 2). In the case of GSH depleted hepatocytes, LDH leakage due to rifamycin SV alone was similar to that of isolated hepatocytes from fed rats and lower than in starved rats, although in both situations (fed and starved) the GSH concentration remained always above 1.5 # m o l / g cells. Similar results were obtained for TBA reactive products arising from the effect of formation due to rifamytin SV and copper (IlL while in the presence of the antibiotic alone there was no considerable evidence of lipid peroxidation. Lipid peroxidation was, however, stimulated by copper (II). This effect is greater in GSH depleted hepatocytes than in cells isolated from fed animals, but with the maximal effect found in the starved hepatocytes in agreement with the observed changes in LDH leakage. In this group of incubations no significant changes were observed in the presence of copper (II) alone on cell viability. Thus, in the presence of the metal (0.05 mM) GSH concentration was 2.0 + 0.6 # m o l / g cells. (6), LDH leakage 53 + 8 #mol/min per g (4) and MDA 250 + 57/tmol/g cells (4). Discussion

Interest in rifamycin SV as a useful therapeutic agent has arisen because of its observed clinical usefulness against viral, inflammatory and immunological response disturbances [9-11]. Quinone containing antibiotics exhibit a wide range of therapeutic spectrum against

several malignant diseases [1]. However, these drugs have also toxic side effects which limit their clinical applications. The precise molecular mechanism of their cytotoxicity is still unknown although free radical production and lipid peroxidation have been suggested [40]. The quinone moiety of these xenobiotics undergoes reversible oxidation-reduction and forms semiquinone and oxygen radicals. Some quinones undergo addition or substitution reactions with nucleophiles such as GSH to form respective glutathione-quinone adducts [41]. Transition metal ions are also important in the induction of both therapeutic and cytotoxic effects. Metal-binding properties of quinone containing drugs are well established [42] as well as the role of metals as catalytic agents in quinone induced redox cycling and free radical production [40]. A plausible explanation for a whole range of oxidative phenomena including cell damage carried out by redox cycling drugs is provided by the free radical induced toxicity hypothesis. In situations in which natural antioxidant mechanisms are overwhelmed as a result of drug overdose or nutritional deficiencies, uncontrolled free radical production may lead to an oxidative degradation of important macromolecules such as nucleic acids, proteins and carbohydrates as well as membrane lipid peroxidation [43-45]. Although relatively high concentrations were used in our study it must be pointed out that rifamyein SV and its derivatives administered to volunteers have been shown to penetrate tissues and to persist at measurable levels in plasma for 24 h after a single oral dose. Peak concentrations of 0.2-0.5 ttg/ml in plasma are easily achieved in man 4 h after 75-300 mg doses and at 24 h or more concentrations are still 0.25 lag/ml [46,47]. Similar concentrations as the ones used in our study have been used by other authors to show the oxidative damage of DNA induced by rifamycin SV [17]. The results obtained in this study show that the effect of rifamycin SV and transition metal ions on metabolic performance and cell viability of isolated hepatocytes under different experimental conditions, i.e, fed, starved and GSH depleted, is due to induction of cell damage in all the three groups of cell populations. The cell susceptibility towards the effect of rifamycin SV depends upon both GSH levels and nutritional status of the cells. The role of metal ions in the induction of the observed effects due to rifamycin SV indicated an increase in the observed effects. The increase in glucose and lactate production by isolated hepatocytes from fed rats in the presence of increasing concentration of rifamyein SV suggests an increase of glucogenolysis and glycolysis which was further stimulated by copper (II). With higher rates of glycogenolysis, in the presence of copper (!I), there is a decrease of pyruvate concentration while lactate increases. Cytosolie redox state was more reduced in these incubations and similar changes were oL,served in the

334 mitochondria. The enhanced glucose release may be related to the induced oxidative stress and represents an energy demanding situation for the cells. The importance of metabolic substrate supply, notably carbohydrates, in the generation of reducing equivalents required by tissue antioxidant processes has been widely established and recently reviewed [48]. The effect of rifamycin SV and heavy metals on gluconeogenesis resulted in an inhibition of this process. in hepatocytes from starved rats low concentrations of rifamycin SV induced a 30~ inhibition of gluconeogenesis. This effect was enhanced by iron (I1) to a 50% inhibition and was maximal in the presence of copper (il) which strongly inhibited glucose production by 77~, Maximal effect on gluconeogenesis was accompanied by a shift of the cytosolic NAD+/NADH couple in the direction of reduction (Table IIl). A possible explanation for this shift is the hydrolysis of ATP. The equivalent rise of phosphate during the decrease of ATP is known to shift the equilibrium of the glyceraldehyde3-phosphate dehydrogenase system, which in turn, is in equilibrium with the lactate/pyruvate couple, towards reduction [49]. The significant decrease in the level of ATP observed in fed and starved heptocytes in the presence of rifamycin SV and copper (II), may be due to its utilization in maintenance of redox-cycling metabolism a n d / o r its oxidative destruction. Although the observed changes may represent a metabolic consequence of the cytotoxic effect induced by rifamycin SV, the possibility that glycogen, glucose and ATP degradation by free radical production arising from redox cycling of the quinone moiety cannot be ruled out. This effect has been reported to occur during oxidative stress [45]. The observed decrease in the GSH concentration could be correlated ~:dth LDH leakage in hepatocytes incubated with rifamycin SV and copper (11), although, even in the presence of the highest antibiotic concentration used, the release of TBA reactive material was not affected (Table If). This depletion in GSH level may be flue to the fact that most xenobiotics a n d / o r their metabolites undergo enzymatic conjugation with cellular GSH through the action of glutathione-S-transfetuses. GSH depletion may also be a consequence of free radical attack during quinone redox cycling with the tripeptide acting as an efficient antioxidant and free radical scavenger. However, the effect of catalase suggests the extracellular production of hydrogen peroxide a n d / o r other reactive species during rifamycin SV oxidation. Thiyi and hydroxyl radicals generated during copper (II) catalyzed cysteine autoxidation were found to cause a decrease in the level of GSH and ATP concentration and a significant increase of LDH leakage in rat hepatocytes [50]. In the present investigation an increase of both LDH leakage and TBA reactive material

was observed after incubation of isolated hepatocytes in the presence of rifamycin SV and transition metal ions. This effect was observed with significant differences especially in those cells isolated from starved or GSH depleted rats and was stimulated mainly in the presence of copper (II). Oxygen consumption by isolated hepatocytes is increased by rifamycin SV and this effect is also stimulated by the addition of copper (If) (Table V). These results together with the partial protection against rifamycin SV cytotoxicity and oxygen consumption achieved by catalase (but not by superoxide dismutase; data not shown) supports a role for hydrogen peroxide, formed extracellularly, as an intermediate of the observed ~ffects although the formation of hydroxyl radicals cannot be ruled out. Other investigations with rifamycin SV have also found TBA-reactive material observed as a result of DNA degradation [17]. In addition, the correlation observed between GSH depletion and LDH leakage shows some characteristics which clearly depend not only on the cell GSH threshold but also on the nutritional status of the hepatocytes. Indeed, cytotoxicity induced by rifamycin SV in terms of GSH depletion, LDH leakage and TBA-reactive material, is greater in both these situations when compared with hepatocytes from fed rats. The depletion of GSH renders the cell more susceptible to peroxidation of membrane lipids leading to cell death [51]. However, the fact that the observed cytotoxicity was greater in starved rats than in GSH depleted cells is an indication of the existence of other factors in the induction and protection against oxidative stress induced cell damage. Acknowledgements G.T.S. dedicates ti~is work to the memory of late friend Professor Dr. Antonio Jordfi Vails. The work was supported by a Grant of the Comisi6n Asesora (PA 86-0169 and PM 89-0095). G.T.S. also thanks the Spanish Ministry of Education and Science (Plan de formaci6n, perfeccionamiento y movilidad del personal investigador 1987) and the British Council for supporting his stay at the Biotechnology Centre, Cranfield Institute of Technology.

References l Nohl, H., Jordan, W. and Youngman, R.J. (1986) Adv. Free Rad. Biol. Med. 2, 211-279. 2 Bachur, N.R., Gordon, S.L. and Gee, M.V. (1978) Cancer Res. 38, 1745-1750. 3 Bannister, J.V. and Thornalley, P.J. (1983) FEBS Lett. 157, 170172. 4 Sies, H., Brigelius, Wefers, H., Muller, A. and Cadenas, E. (1983) Fund. Appl. Toxicol. 3, 200-208. 5 Meyers, C.E., McGuere, W.P., Liss, R.H., lfrim, 1., Grotzinger, K. and Young, R.C. (1977) Science 197, 165-157.

335 6 Doroshow, J.H. and Hochstein, P. (1982) in Pathology of Oxygen (Autor, A.P., ed.), pp. 245-259, Academic Press, New York. 7 Sensi, P., Maggi, N., Furesz. S. and Maffi, G. (1982) in The Use of Antibiotics (Kucers, A. and Bennet, N.M., eds.), pp. 547-551, Heinemann, London. 8 Wehrli, W. and Staehelin, M. (1975) in Mechanism of Action of Antimicrobiai and Antitumor Agents. Antibiotics III (Corcoran, J.W. and Hahn, F.E., eds.), pp. 252-268, Springer-Verlag, Berlin. 9 Engle, C.G., Lasinski, E. and Gelzer, J. (1970) Nature 228, 11901191. 10 Caruso, F., Montrone, F., Fumagalli, M., Parono, C., Santandrea, S. and Gendini, G. (1982) Ann. Rheum. Dis. 41,232-236. 11 Kasik, J.E. and Monick, M. (1981) Antimicrob. Agents Chemother. 19, 134-138. 12 Wehrli, W. and Staehelin, M. (1971) Bacteriol. Rev. 35, 290-309. 13 Lowder, J.F. and Johnson, R.S. (1987) Biochem. Biophys. Res. Commun. 147, 1129-1136. 14 Lester, W. (1972) Anno, Rev. Microbiol. 26, 85-102. 15 Scrutton, M.C. (1977) FEBS Lett. 78, 216-220. 16 Kono, Y. (1982) J, Biochem. 91,381-395. 17 Quinlan, G.J. and Gutteridge, J.M.C. (1967) Biochem. Pharmacol. 36, 2629-2633. 18 Jacoby, W.B. (1977) Adv. Enzymol. 46, 381-412. 19 Jones, D.J., Eklow, L., Thor, H. and Orrenius, S. (1981) Arch. Biochem. Biophys. 210, 505-516. 20 Sies, H. (1985) Oxidative Stress, Academic Press, London. 21 Meister, A. (1988) J. Biol. Chem. 263, 17205-17208. 22 Anundi, I., Hogberg, J. and Stead, A.H. (1979) Acta Pharmacol. Toxicol. 45, 45-51. 23 Shez, O.T., Pallardo, F., Romero, F.J. and Viha (1985) Biochem. Pharmacol. 34, 453-454. 24 Sfiez, G.T., Romero, F.J. and Viha, J. (1985) Arch. Biochem. Biophys. 241, 75-80. 25 Wendel, A., Fuerstein, S. and Konz. K.H. (1979) Biochem. Pharmacol. 28, 2051-2055. 26 Romero, F.J. and Viha, J. (1983) Biochem. Educ. 11,135-136. 27 Berry, M.N. and Friend, D.S. (1969) J. Cell. Biol. 43, 506-520. 28 Jordfi, A.. Siiez, G.T., Portol6s, M., Pallard6, F.V., Jimenez Nacher, J. and Gasc6, E. (1988) Biochemie 70. 1417-1421. 29 Boyland, E. and Chasseaud, L.F. (1967) Biochem. J. 104, 95-102. 30 Griffith, O.W. and Meister, A. (1979) J. Biol. Chem. 254, 75587560. 31 Slein, M. (1974) in Methods of Enzymatic Analysis (Bergmeyer. H.U., ed.), pp. 117-123, Academic Press. New York.

32 Gutman, I. and Wahlefeld, A.W. (1974) in Methods of Enzymatic Analysis (ergmeyer, H.U., ed.), pp. 1464-1468, Academic Press, New York. 33 Czok, R. and Lamprecht, W. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H.U., ed.), pp. 1446-1451. Academic Press, New York. 34 Mellamby, J. and Williamson. D.H. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H.U.. ed.), pp. 1836-1839, Academic Press. New York. 35 Williamson, D.H. and Mellamby, J. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H.U., ed.), pp. 514-527, Academic Press, New York. 36 Williamson, D.H., Lurid, P. and Krebs. H.A. (1967) Biochem. J. 103, 514-527. 37 Brigelius, R., MuckeL C., Akerboom. T.P.M. and Sies, H. (1983) Biochem. Pharmacol. 32, 5529-5534. 38 Vi~a, J. Shez, G., Wiggings, D., Roberts, A.F.C., Hems, R. and Krebs, H.A. (1983) Biochem. J. 212, 39-44. 39 Stacey, N. and Priestley, B.G. (1978) Toxicol. Appl. Pharmacol. 45, 41-48. 40 Powis, G. (1989) Free Rad. Biol. Med. 6, 63-101. 41 Finley, K.T. (1974) in The Chemistry of Quinoid Compounds. Part 2 (Potil, S., ed.), pp. 877-1144, Wiley. London. 42 Albert, A. and Rees, C.W, (1956) Nature 177, 433-434. 43 Slater, T.F., Cheesman, K.H., Davies, M.J., Proudfoot. K. and Xin, W. (1987) Proc. Nutr. S ~. 46, 1-12. 44 Gower, J.W. (1988) Free" l, ad. Biol. Med. 5, 95-111. 45 Sies, H. (1986) Angew. Chem. 25, 1058-1071. 46 Anand, R. and Moore, J. (1986) The Lancet i, Jan. 1 t. 97-98. 47 Mozzi, E., Geminiani, R., Cantaluppi, G., Marchetti. V., Veltaro, M.P. and Sandi, A. in Proceedings of 13th International Congress of Chemotherapy, Vienna, Aug. 28-Sept. 2. 1983. 48 Godin, D.V. and Wohaieb, S.A. (1988) Free Rad. Biol, Med. 5. 165-176. 49 Krebs. H.A. (1971) in Regulation of GLuconeogenesis (Soling, H.D. and Willms, B.. eds.), pp. 114-117, Academic Press. New York. 50 Shez, G.T., Thornalley, P.J., Hill, H.A.O., Hems. R. and Bannister. J.V. (1982) Biochim. Biophys. Acta 719, 24-31. 51 Nicotera, P. and Orrenius. S. (1986) Adv. Exp. Med. Biol 197. 41-51.

Effect of metal ion catalyzed oxidation of rifamycin SV on cell viability and metabolic performance of isolated rat hepatocytes.

The effect of rifamycin SV on metabolic performance and cell viability was studied using isolated hepatocytes from fed, starved and glutathione (GSH) ...
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