Cytotoxicity in pig hepatocytes induced by 8 ...

vet. Pharmacol. Therap. 23, 37-44, 2000,

TOXICOLOGY

Cytotoxicity in pig hepatocytes induced by 8-quinolinol, chloramine-T and natamycin

.

M. R. MARTÍNEZ-LARRAÑAGA A.ANADÓN

M. L. FERNANDEZ-CRUZ

Martínez-Larrañaga. M. R., Anadón, A.. Femandez-Cruz. M. L., Díaz. M. J., Martínez, M. A., Frejo, M. T., Martínez, M., Tafur, M. Cytotoxicity in pig hepatocytes induced by 8-quinolinol. chloramine-T and natarnycin. J. vet. Pharmacol. Therap. 23, 37-44.

M. J. DÍAZ M. A. MARTINEZ M. T. FREJO M. MARTÍNEZ & M. TAFUR Department 01 Toxicology and Pharmacology. Faculty 01 Veterinary Medicine. Universidad Complutense de Madrid. 28040 Madrid. Spain

The potential cytotoxic effects of the compounds 8-quinolinol. chloramine-T and natarnycin have been studied in isolated pig hepatocytes. The relative cytotoxicity of these compounds was evaluated on the basis of the leakage of cytosolic lactate dehydrogenase (LDH) , 3-(4,5 dimethyl)thiazol-2-yl.-2.5-diphenyl tetrazolium bromide (MTT) reduction by mitochondrial dehydrogenases, uptake of neutral red (NR) by cytosolic lysosomes, glutathion (GSH) depletion and oxidized glutathion (GSSG) effiux after 24 h exposure. Evaluation of the 20%, 50% and 80% reduced absorbance data obtained from the parameters NR 20 , NR so , and NR so , and MTT 20 , MTT so and MTI'so enabled us to rank these compounds in decreasing order of cytotoxicity: 8-quinolinol > natamycin > chloramine-T. AIso for the parameters LDH and GSH, chlorarnine-T appears to be less cytotoxic than natamycin and 8-quinolinol. Our study demonstrated that pig hepatocytes may be a useful model for examining cytotoxic events of drugs to be used in pigs. therefore avoiding possible extrapolation problems due to species differences. (Paper received 7 May 1999; accepted for publication 23 November 1999) A. Anadón. Department of Toxicology and Pharmacology, Facu1ty of Veterinary

Medicine, Universidad Complutense de Madrid. 28040 Madrid. Spain. E-mail: [email protected]

INTRonUCTION Fungicidal and bactericidal agents are among the most commonly used therapeutic drugs. Chemically. they are a heterogeneous group of compounds. In this study. we evaluated the cytotoxicity of three compounds. 8-quinolinol. chloramine-T and natamycin (pimaricin) (Fig. 1). The compound 8-quinolinol possesses antibacterial. antifungal and antiprotozoal activity. It is used as a topical antiseptic and has a variety of industrial applications and as a preservative in cosmetics (Ackart et al .• 1975; Gershon et al .• 1991; Khan et al .• 1994). Chloramine-T. a byproduct of saccharin manufacture. is employed as an antiseptic. biocide and is a constituent of many pharmaceutical preparations. AIso. chloramine-T is widely used as a disinfectant for plant sterilization in the food industry and for the disinfection of drinking water (Van Gils. 1970; Andres. 1982; Hegna & Clausen. 1988). Natamycin. a tetraene polyene antibiotic. has been used as a topical therapeutic in animals and humans for over 30 years. and it is also used as an antifungal agent in food processing. It is authorised in the European Union as a food additive for surface treatment of certain cheese and sausages. Natamycin appears to be an extremely effective inhibitor of ©2000 Blackwell Science Ltd

mould growth and mycotoxin production (Klis et al .• 1959; Newcomer et al.• 1960; Dittrich & Sponholtz. 1981; Ray & Bullerman. 1982; Gourama & Bullerman. 1988; Verma et al .• 1988; Reuben et al .• 1989; n'Urso et al .. 1990; Hoppe et al .• 1995). The compounds 8-quinolinol. chloramine-T and natamycin are added to many foods as preservatives in order to suppress bacterial and mould growth and mycotoxin production. Although some aspects of the bactericidal and fungicidal properties have been reported in the scientific literature. limited information is available on the toxic actions of these compounds. The potential antifungal usefulness of 8-quinolinol. chloramineT and natamycin in feedstuffs for pigs and other domestic animals requir~s information on toxicological profile to further define the use safety of these active substances. Hepatocyte-based systems are widely used to study the toxic properties of xenobiotics in vitro (Fry & Bridges. 1979; Ratanasavanh et al., 1988). Previous studies have proven the usefulness of cultured pig hepatocytes in the evaluation of biotransformation and cytotoxicity of severa! drugs that are known to be hepatotoxic in vivo in mammals (Zimmerman. 1979; Hoogenboom et al., 1989. 1990.1991; Monshouwer et al.. 1996; Mengelers et al .• 1997). We have therefore investigated the cytotoxicity of the com37

38 M.R. Martínez-Larrañaga et al. Chemical structure

Compound

'ro

8-Quinolinol. sulfate (2: 1) (sall) (CtH,NOl2H,So.

Chloramine-T (C,HrCl N NllÚ)S)

::--..

«-

.., 2038 in male rals (NTP.1991)

388.40

......-:

rO

227.67

>2000 in male rals (unpublished data oblained in our laboralory)

SOzNQlb

2730 in male rats 665.75

Nalamycin (CIltt.,NOI)

4670 in female rats

Flg. l. Comparison 01 the pathological and chemical properties of three antibacterial and antifungal compounds.

(Sudavari el al. 1989)

pounds 8-quinolinol. chloramine-T and natarnycin using this in vJtra model. In this study. we evaluated the cytotoxicity of 8-qulnolinol. chloramine-T and natamycin using 24 h exposure of primary cultures of pig hepatocytes. Cytotoxicity was measured by evaluating reduction of 3-(4.5-dimethylthiazol2yl)-2.5-diphenyl- tetrazolium bromlde (MTT). neutral red uptake (NR). lactate dehydrogenase leakage (LDH) and glutathione (GSH) depletlon.

MATERIALS ANV

M~THUVS

Chemicals

Sodium chloride (NaCl). potassium chloride (KCl) , Titriplex Vi (EGTA), calcium chloride dihydrate (CaCh.2H 2 0), sodium bicarbonate (NaHC0 3 ), 1-fluoro-2,4-dinitrobenzene, glacial acetic acid, ethanol absolute, monopotassium phosphate, dipotassium phosphate, perchloric acid 7(}-72%, sodium acetate trihydrate, each of analylical grade, were obtained from Merck (Dannstadt, Genoany). Hepes, bovine serum albumino Hank's balanced salt solution, Williams'medium E, minimum essential medium Eagle (EMEM medium), L-glutamine, collagenase type 1, trypan blue solution 0.4%, foeta! calf serum, insulin (1 mgjmI..) , 5000 units penicillin and 5 mg streptomycin/mI.. solution, 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyl-tetrazolium bromide (MTT), Triton X-lOO, iodoacetic acid, 8-quinolinol sulfate 2:1 (salt), chloramine-T (N-chloro-ptoluene-sulfonamide sodium salt), natamycin (pimaricin), pyruvic acid and I3-nicotinamide adenine dinucleotide reduced fono (13NADH) , neutral red test kit. Dulbecco's phosphate buffered saline (PBS), glutathione reduced fono (GSH) and glutathione disodium salt oxidized fono (GSSG) were obtained from Sigma Chemical Co. (St l.ouis, USA). Methanol. glacial acetic acid and isopropanol. each for HPLC grade were supplied by Sharlau (Barcelona, Spain). Water was from a Millipore. Alpha-Q, Ultrapure Water System (Millipore S.A., Malsheim. France).

Animals

The experiments were perlormed using livers 01 tour clinically healthy male pigs (Landrace x Large White), weighing 100 kg from a breeding farm. The pigs were placed in individual pens provided with facilities for drinking (ad libitum) and eating (0.75 kg of pelleted, antibiotic-free food twice daily). To acclimatize them to their environment the plgs were placed in pens 7 days prior to the experimento The animal house was maintained at room temperature (25 ± 2 oC) and at 45-65% relative humidity. The pigs were killed by electrical stunning. followed by immediate bleeding and a liver sample (a portion of the left laterallobe) weighing about 100 g was taken from each animal. Animal use in this study was conducted in accordance with Complutense University animal house guidelines for the care and use of laboratory animals. The experimental protocol and procedures were approved by the Spanish State Commission for Laboratory Animals.

lsoIation and culturing aJ hepatocytes

Hepatocytes were prepared for monolayers from the liver sample by a three-step perfusion technique as described previously by Hoogenboom et al. (1991). The liver sample was perfused with 1000 mI.. of buffer I (8.3 g NaCl, 0.5 g KCl, 2.4 g Hepes, 0.19 g EGTA, pH 7.6) followed by perfusion with 1000 mI.. of buffer II (buffer I without EGTA) and subsequently by perfusion with 300 mI.. of a third buffer (3.9 g NaCI. 0.5 g KCl, 24 g Hepes and 0.70 g CaCI 2 .2H2 0, pH 7.6) containing 0.05% collagenase under recirculation for 30 mino During the perfusion, buffers were gassed with carbogen (95% O2 , 5% C0 2 ). After digestion, hepatocytes were washed with ice-cold Hank's balanced salt solution containing 0.2% bovine serum albumino Cells were collected by centrifugation at 50 x g, 4 oC for 4 mio and were suspended in Williams' medium E. An aliquot of the final cell suspension was used to determine the number and fraction of \Q¿UUU

B1aCKWell :sclence

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Cytotoxicity by 8-qulnolinoI. chloramine-T and natamycin 39

trypan blue-excluding cells as described by Jauregui et al. (1981). Initial cell viability determined by trypan blue exclusion was 93 ± 4%. Cells were diluted up to 1.25 million or 0.2 million/mL ofWilliams'medium E. Cells, 3.125 million in 2.5 mL ofmedium were seeded in uncoated 60-mm culture dishes or 0.04 million in 200 IlL ofmedium E were seeded in 96-well culture plates and allowed to attach for ~ before the incubation medium was renewed. Routinely, cells were incubated and exposed in Williams'E medium supplemented with 5% foetal calf serum, 0.05% insulin, 1% penicillin/streptomycin solution, at 37°C under an atmosphere of 95% air/5% CO 2 •

Exposure

01 cells

After 1 day in culture, the hepatocytes were exposed to 8quinolinol (1, S, 10, 25, SO, 100, 350 and 700 11M), to chloramine-T (1, 10, 100, sao, 800, 1000. 2500. 5000 and 10000 11M), and to natamycin (1, 2.5, S, 8. 10, 25. SO, 80. 100 and 2 SO 11M) for 24 h. Hepatocyte monolayers were treated with these test compounds dissolved in EMEM medium (containing 0.03% glutamine) for MTT and neutral red assays and in Williams' medium E (without foetal calf serum, insulin, penicillin and streptomycin) for lactate dehydrogenase and glutathione assays. In each experiment. for MTT and neutral red assays, eight wells in 96-well plates (100 IlL/well) were exposed for each data point; for lactate dehydrogenase and glutathione assays. five dishes (2.5 mL/dish) were treated for each data point.

Cytotoxicity assays MTT and neutral red The MTT reduction and the neutral red uptake were measured according to Mosmann (1983) and Borenfreund & Puerner (1985), respectively. Medium was removed from the 96-well plates and 100 IlL of EMEM medium containing MTT (500 Ilg/ mL) or neutral red (330 Ilg/mL) was added to each well. MTT medium was replaced after incubation for 4 h with isopropanol (100IlL/well). Neutral red containing medium was removed after 4 h incubation, the cells washed with PBS and destain (lOOIlL, 1% glacial acetic in 50% ethanol) was added to each well. Absorbance of each well was read at 540 nm or 570 nm (neutral red or MTT, respectively) uSing a Bio-Rad Model 550 microplate reader (Bio-Rad Laboratories. Hercules, CA. USA).

Lactate dehydrogenase (LDH) WH activity in the medium and cells was measured as described by Jauregui et al. (1981). Intracellular enzyme was released by Iysing the cells in phosphate bulTered saline containing 1 % Triton X-lOO. WH activity was assayed by the pyruvate to lactate reaction, monitoring the rate of absorbance decrease at 340 nm and 37°C. The substrate composition was 1.4 mM pyruvate. 60 mM monopotassium phosphate. 60 mM dipotassium phosphate and 10 mM NADH (pH 7.45). Activity is expressed in international units (IU)/mg cell protein. Percentage of WH leakage was defined as IU WH/mg cell protein in medium ©2000 Blackwell Science Ltd. J. veto Pharmacol. Therap. 23. 37-44

divided by total IU LDH/mg cell protein x 100%. Total LDH is the addition of intracellular and tissue culture medium LDH expressed in IU /mg cell protein. Protein in cells and medium was measured by the method ofLowry et al. (1951), as modified by Peterson (1977).

Glutathione Reduced glutathione (GSH) and oxidized glutathione (GSSG) were determined by an HPLC procedure based upon the initial formation of S-carboxymethyl derivatives of free thiols with iodoacetic acid followed by conversion of free amino groups to 2,4-dinitrophenyl derivatives by reaction with 1-fluoro-2,4dinitrobenzene as described by Reed et al. (1980). Preparation 01 hepatocyte lractions. Hepatocytes, 1.25 million cells/mL, were washed once in saline. The cell pellet was resuspended in 1 mL of 0.9% saline, 0.05 mL of 70% perchloric acid was added. and the protein removed by centrifugation at SO x g 4 oC for 4 mino A 0.5-mL aliquot of the supernatant was treated immediately with 50 IlL of a fresh aqueous solution (4 Ilmol) iodoacetic acid and then neutralized with an excess of NaHCO 3 (dried powder). After 60 min in the dark at room temperature (25 ± 2 oC), 0.5 mL of an alcoholic solution of 1fluoro-2,4-dinitrobenzene (1.5 mL/98.5 mL absolute ethanol) was added and the reaction allowed to proceed for 4 h in the dark. After derivatization. the samples were stored in the dark at 0-5 oC, for no more than 2 weeks. until HPLC analyses. Determination 01 GSH and GSSG derivatives by HPLC. The N 2.4-dinitrophenyl (DNP) derivatives of GSH and GSSG formed were determined on the HPLC system (Kontron Instruments 325 system with a Kontron Instruments HPLC 332 detector and a Kontron Instruments PC Integration Pack Version 3.90; Kontron Instruments S.p.A., Milano, Italy) equipped with a Teknokroma Supelcosil LC-NH 2 column (5 11m. 250 x 4.6 mm) (Teknokroma, Sant Cugat del Valles, Barcelona, Spain). The mobile phase was a mixture of solvent A and solvent B and a flow of 1 mL/min was used. Solvent A was MeOH: water (4:1 v/v). Solvent B was prepared as follows: 272 g sodium acetate trihydrate, 122 mL water, and 378 mL glacial acetic acid were mixed and 200 mL of the resulting added to 800 mL of solvent A. Derivatives of GSH and GSSG were eluted using an isocratic period of 10 min followed by a gradient started at 25% and programmed linearly to 95% solvent B over a 1ü-40-min periodo Chromatography was performed at 25 ± 2 oC with detection at 365 nm. Peak areas in the sample chromatograms were quantitated by use of the external standard technique using GSH and GSSG reference standards under assay conditions used for biological samples. Typical retention times: GSH-derivative, 30 min; GSSG-derivative, 36 mino Protein was measured as described above.

Statistical analysis Each experiment was performed at least five times, uSing five dishes or eight wells for each concentration of test agent and the mean values were calculated and used to construct the doseresponse cytotoxicity data. AlI dose-response cytotoxicity data

40 M.R. Martínez-Larrañaga et

were presented as means ± SEM from four separate hepatocyte isolations from four liver samples of four pigs. Dose-cytotoxic response curves were constructed using the Microcal Origin 3.46 computer program (Microcal Software Inc.. Northampton. MA. USA). For each drug. the concentrations needed to reduce absorbance of the NR or MTI by 20% (NR20 and MTI20 values. respectively). 50% ~so and MTI so . respectively) and 80% (NRso and MTIso• respectively) were determined from the dose-response curve. Unpaired Student's t-tests were performed. in appropiate comparisons with controls. A P-value < 0.05 was considered to be statistically significant.

in pig hepatocyte cultures was also observed (Table 2). For natamycin. a 50-11M concentration was required to cause a significant LDH release after 24 h of exposure. However. for 8quinolinol and chloramine-T 350 11M and 800 11M concentrations. respectively. were required to cause a significant LDH release after 24 h of exposure. Parameters which measure metabolic competence of the cells were altered by 8-quinolinol. chloramine-T and natamycin (Table 2). GSH was significantly reduced and GSSG effiux was significantly increased in cells exposed to 50 and 80 11M natamycin. 50. 100 and 350 11M 8-quinolinol. and 800 and 1000 11M chloramine-T. after 24 h of incubation.

RESULTS DISCUSSION Figures 2 and 3 illustrate the dose-response cytotoxicity. as determined by MTI reduction and neutral red uptake. of 8quinolinol. chloramine-T and natamycin in the pig hepatocyte primary culture system. Natamycin at 1-25 11M. 8-quinolinol at 1-25 11M and chloramine-T at 1-100 11M had no etTect on cell viability. A significant decrease in MTT reduction was seen after 24 h in pig hepatocyte monolayers with 50-250 11M natamycin. 50-700 11M 8-quinolinol and 500-10000 11M chloramine-T. Similarly. a significant reduction in neutral red uptake was seen after 24 h in pig hepatocyte monolayers with 50-700 11M 8quinolinol. 50-250 11M natamycin and 500-10000 11M chloramine-T. On the basis of initial and midpoint cytotoxicity (MTT 20 • NR 20 and MTT so . NR so . respectively) values (Table 1). the sequence of potency in decreasing order for these compounds was: 8-quinolinol > natamycin > chloramine-T. with no marked ditTerences in sensitivity between NR 20 and MTT 20 for 8-quinolinol and natamycin. On the other hand. a dose-response relationship for 8quinolinol. chloramine-T and natamycin-induced LDH leakage

Because mycotoxins produced by many moulds which contaminate foods. a list which becomes longer each day. are causing important implications in human and animal health. the development of antifungal and antimycotic agents has been a goal of the pharmaceutical and food industries. Compounds such as 8-quinolinol. chloramine-T and natamycin are receiving attention as posible antifungal agents in feedingstutTs for pigs and other domestic animals. Because the toxic etTects of these compounds have been poorly characterized. as an initial step we evaluated the general cytotoxicity of 8-quinolinol. chloramine-T and natamycin at ditTerent concentrations in pig hepatocytes in order to select toxicologically relevant concentrations of these compounds for further studies. The leakage of cytosolic LDH. MTT reduction by mitochondrial dehydrogenases. neutral red uptake by cytosolic Iysosomes and GSH depletion were utilized as markers for cytotoxicity. The toxicity induced by 8-quinolinol. chloramine-T and natamycin on the parameters MTT reduction. NR uptake. LDH

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l'ig. Z. Uose-response curves lar MTI reduction (% oC control) from monolayer pig hepatocytes exposed Cor 24 h to the Collowing compounds: 8-quinolinol. chloramine-T and natamycin. Each curve is composed oC means ± SEM oC Cour experiments.

J. veto Pharmacol. Therap. 23. 37-44

Cytotoxicity by 8-quinolinol. chloramine-T and natamycin 41

o chloramine - T • natamycin

.--.

-8e

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Fig. 3. Dose-response curves for neutral red Z uptake (% of control) from monolayer pig hepatocytes exposed for 24 h to the following compounds: 8-qulnollnol. chloramine-T and natamycin. Each curve is composed of means ± SEM oC four experiments.

40 20

1

leakage and GSH depletion was concentration-dependent (Figs 2 and 3. and Table 2). Leakage of the cytoplasmic enzyme LDH is commonly used as an index of hepatocellular damage and is applicable to both hepatocyte suspensions and monolayer cultures. LDH functions in the last step of glycolysis. catalyzing the reversible reduction of pyruvate to lactate (Lehninger. 1972). As LDH is released by hepatocytes in distress. it has been assayed during drug toxicological studies as activity in either supernatant media or in cells in response to drug concentration and time of exposure (Dujovne et al .. 1972: Anuforo et al .• 1978: Tolman et al .. 1978). LDH leakage levels were significantly higher at concentrations equal or higher than 50 llM natamycin. 350 11M 8-quinolinol and 800 11M chloramine-T. These findings were supported by the MTT reduction and NR uptake data. A significant decrease in MTT reduction and NR uptake was seen with concentratlons equal or higher than 50 11M natamycin. 50 11M 8-quinolinol and 500 11M chloramine-T. For chloramine-T and with marked difference for 8-quinolinol. cytotoxicity was detected with lower concentratlon in the MTT reduction and NR uptake than in the LDH leakage. LDH leakage is indicative of plasma membrane stability. while MTT reductlon and NR uptake are indicative of mitochondrlal and Iysosomal integrity of viable cells. respectively. We also investigated the effect of 8-quinolinol. chloramine-T and natamycin on GSH depletion and GSSG production. GSH is an essential intracellular reductant for maintenance of thiol groups on intracellular proteins and for antioxidant molecules such as ascorbate and ex-tocopherol. GSH pro vides protection to cells against oxldative stress and other types of damage (Acosta et al.. 1978: Larsson et al .• 1983: Toussaint et al .• 1993). When cellular GSH is reduced or depleted. the physiological formation of reactive oxygen species is not largely opposed and cellular damage may occur. GSH depletion can be achieved either by inhibiting GSH synthesis (Griffith & Meisser. 1979: Remoro & ©2ooo Blackwell Science Ltd.l. veto Pharmacol. Therap. 23. 37-44

100

10

1000

10000

Concentration (iJm) Table l. Comparative cytotoxicity of 8-quinolinol. chloramine-T and natamycin. as determined by the neutral red (NR) and tetrazolium M'IT assay 8-Quinolinol NR assay

Mean ±SEM

NR 20 43.0 46.3 46.4 47.3 45.7 ±0.9

NR so 57.7 58.9 51.4 54.4 55.6 ±1.7

M'IT assay

NRso 77.4 74.9 56.7 62.9 68.0 ±4.9 Natarnycin

M'lT2o

M'lTso

M'lTso

54.9 55.5 51.9 53.2 53.9 ±0.8

64.4 63.9 63.1 58.7 62.5 ± 1.3

75.5 73.6 76.5 64.8 72.6 ±2.7

NR assay NR 20 59.7 49.8 41.3 42.2 Mean ±SEM

48.2 ±4.3

MTT assay

NR so NRso 86.2 124.5 83.9 141.3 71.9 124.9 83.1 163.4 81.3 138.5 ±3.2 ±9.2 Chloramine-T

M'lT20

M'lT so

M'lTso

50.1 55.3 52.1 54.3

81.8 97.6 81.4 87.0

133.6 172.0 127.3 139.4

52.9 ±1.2

86.9 ±3.8

143.1 ±10.0

NR assay NR20 758.3 689.7 836.3 817.7 Mean ±SEM

755.5 ±33.1

NRs o 1105.9 1116.4 1178.2 1208.6 1152.3 ±24.7

MTT assay NRso 1612.8 1807.1 1659.8 1786.2

M'lT20 824.4 795.2 839.1 881.7

1716.5 ±47.5

835.1 ±18.0

Ml'f so 1135.4 1183.6 1247.1 1240.9 1201.7 ±26.4

M'lT so 1563.7 1761.6 1832.6 1746.1 1726.0 ±57.3

Values are expressed as mean concentrations (f.IM) ± SEM. from four experiments, required to reduce absorbance by 20% (NR20 and M'lT20), by 50% (NR so and M'lT so ) and by 80% (NRso and M'lTso). The compounds are Usted accordlng to their NR 20 values.

42 M.R. Martínez-Larrañaga et al. Table 2. E1Tects of 8-quinolinol. chloramine-T and natarnycin on IDH leakage. GSH depletion and GSSG production in pig hepatocytes IDH (IU/mg cell protein) Medium (x 0.001)

Test agentt (¡.tM)

Control Natamycin 1 2.5

S 8 10 25

so 80 100 Control 8-Quinolinol

10 25

SO 100 350 700 Control Chloramine-T 1 10 100 500 800 1000 2500

.,

20.0

± 1.6

21.0 ± 1.7 20.5 ± 1.7 20.0 ± 1.2 21.7 ± 1.4 21.0 ± 0.4 23.5 ± 2.2 80.2 ± 1.7'" 125.0 ± 6.5'" 197.5 ± 8.5'" 21.5

± 1.3

19.7 ± 22.7 ± 21.0 ± 20.0 ± 18.7 ± 23.0 ± 105.0 ± 235.0 ± 21.0

1.1 1.6 0.9 1.2 1.0 0.7 10.4" 13.2··'

± 0.9

21.5 ± 0.6 19.5 ± 0.3 21.7 ± 1.4 20.5 ± 0.6 30.0 ± 0.7'·' 33.7 ± 0.7·" 277.5 ± 7.5'"

0.32

± 0.01

5.9

0.31 ± 0.01 0.31 ± 0.01 0.31 ± 0.01 0.32 ± 0.01 0.32 ± 0.01 0.33 ± 0.01 0.29 ± 0.01" 0.25 ± 0.01*** 0.25 ± 0.01*** 0.32

± 0.01

± 0.01

54.0

± 0.2

6.7 ± 0.1 6.8 ± 0.2 6.5 ± 0.2 6.5 ± 0.1 5.8 ± 0.1 6.4 ± 0.1 31.1 ± 0.9'·' 55.0 ± 0.6··' 6.2

0.33 ± 0.01 0.31 ± 0.01 0.33 ± 0.01 0.33 ± 0.01 0.29 ± 0.01* 0.28 ± 0.01** 0.23 ± 0.01'"

± 0.1

6.2 ± 0.3 6.2 ± 0.2 6.1 ± 0.1 6.3 ± 0.2 6.1 ± 0.1 6.7 ± 0.6 21.9 ± 0.3'" 33.3 ± 0.7*** 44.0 ± 0.6*** 6.3

0.28 ± 0.01 0.31 ± 0.01 0.30 ± 0.01 0.28 ± 0.01 0.30 ± 0.01 0.33 ± 0.01 0.23 ± 0.01·· 0.19 ± 0.01*** 0.32

GSH (nmol/mg protein) in cells

IDH leakage in cells

Cells (% oC total)

± 0.2

6.1 ± 0.1 5.9 ± 0.1 6.2 ± 0.2 5.9 ± 0.2 9.2 ± 0.2"· 10.6 ± 0.2'" 54.4 ± 0.3'"

± 0.6

52.3 ± 3.2 52.0 ± 2.7 52.9 ± 1.8 51.8 ± 3.3 52.4 ± 3.0 52.3 ± 3.7 37.1 ± 2.8" 34.3 ± 2.5**

GSSG (nmol/mg protein) 3.0 ± 0.1 3.2 ± 0.3 3.0 ± 0.1 3.0 ± 0.2 3.1 ± 0.1 3.1 ± 0.1 3.0 ± 0.1 4.1 ± 0.2" 4.5 ± 0.3"

52.6

± 0.9

3.0

52.5 52.9 53.2 54.9 36.5 41.8 41.3

± 0.8 ± 1.8 ± 1.5 ± 1.9 ± 0.5'*' ± 0.9**' ± 0.7···

3.1 ± 0.1 3.0 ± 0.2 3.1 ± 0.2 3.3 ± 0.2 4.3 ± 0.2'* 4.9 ± 0.4' 5.2 ± 0.3·'·

53.7

± 1.3

2.7

57.1 ± 2.2 55.0 ± 2.1 53.5 ± 1.3 54.2 ± 1.8 44.2 ± 2.2·' 43.4 ± 1.8"

± 0.1

± 0.2

2.9 ± 0.4 2.5 ± 0.3 2.7 ± 0.3 2.6 ± 0.2 5.2 ± 0.1·" 7.6 ± 0.2 '"

tCompounds are listed according to their cytotoxic concentrations. Values are expressed as means ± SEM from four experiments. IDH dehydrogenase; GSH = glutathione. reduced; GSSG = glutathione. oxidized. 'P < 0.05; "P < 0.01; "'P < 0.001

Sieso 1984). inhibiting the glutathione redox cyele (Orrenius et al .• 1983). or by using compounds that are readily conjugated with GSH (Smith et al.. 1983). Intracellular GSH levels may decrease as a result oC GSH acting as a protective agent trapping potential reactive intermedia tes or by acting as a substrate in the detoxification oC hydrogen peroxide by GSH peroxidase leading to the Cormation oC GSSG (Holtzman. 1981; Vroomen et al.. 1987. 1988). The compounds 8-quinollnol and natamycin at concentratlons equal or higher than 50 ~M. and the compound chloramine-T at concentrations equaI or higher than 800 ~M induced a significant GSH depletion and/or GSSG production. Under certain conditions oC metabollc activation. 8-quinollnol is an in vitro mutagen (Talcott et al .• 1976) but is noncarcinogenic to rats and mice (Tennant et al.. 1987). However. 8-quinollnol is structurally somewhat related to carcinogenesis promoters of the teleocidin c\ass (Sugimura. 1982). Both teleocidin and 8quinollnol possess aryl nitrogen atoms and nearby hydroxyl groups. One mechanism could explain GSH depletion observed in our study: biotransCormation oC 8-quinollnol could inelude oxidation to a reactive intermediate which is inactivated by formation of a glutathione. Nevertheless. further experiments are

=

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required to establlsh 8-quinollnol metabolismo Chloramine-T is an oxidizing agent that appears to be Cairly specific Cor methionine and cysteine residues of membrane proteins (Schechter et al .. 1975). These actions are similar to the oxidizing effects oC hydrogen peroxide (Means & Feeny. 1971). The results oC the present study also demonstrated that chloramine-T produced a dose-dependent GSH depletion. Chloramine-T (Na salt oC Nchloro-p-suIConamide) is thought to exist in a complex series oC equilibria in aqueous solution: sodium hypochlorite (hypochlorous acid). protonated chloramine-T. dichloramine-T. and ptoluenesulConamide (Alexander. 1973; Balasubramanian & Thiagarajan. 1975; Campbell & Johnson. 1978). These active species could potentially reduce the levels oC GSH and increase production oC GSSG. Because 8-quinollnol. natamycin and chloramine-T may be used as Cungicidal and bactericidal agents in feedstuffs Cor pigs and other domestic animaIs. and as GSH depleting compounds may adversely affect cellular Cunctions. the identification oC the effective depletion oC GSH in isolated pig hepatocytes by these compounds is oC prime importance to avoid or llmit unnecessary exposure to it. The major limitations oC this research arise when trying to extrapolate these findings to animal ©2000 Blackwell

~cience

Ltd. J. vet. Pharmacol. l'herap. 23. 37-44

Cytotoxicity by 8-quinolino/. c1tloramine-T and natamycin 43

and human exposure. Obviously. the validity of in vitro-in vivo correlations depends on the elimination of aH possible extraneous factors related to the pharmacokinetics of the compound under consideration. Further comprehensive studies of the toxicity of these effective compounds may provide valuable information about their possible wide spread application with no or minimum undesirable side-effects,¡,,¡.,

ACKNOWLEDGMENTS This work was supported by the European Union. Project AIR3CT94-1325. Brussels. Belgium. and by the Comisión Intermirristerial de Ciencia y Tecnología. Project SAF95-1213-CE. Spain. The authors would like to thank Dr R.A. Kuiper of the State Institute for Quality Control of Agricultural Products (RIKILT). Wageningen. The Netherlands. for his hospitality with Dr M.L. FernandezCruz (ESF feHowship) for acquiring knowledge to perform pig hepatocyte preparation.

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