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Adenine and pyridine nucleotide concentrations and relationships to 2,6-dinitrotoluene metabolism in cultured rat liver slices
Pergamon
0887-2333(94)E0026-P
Toxic'. in Vitro Vol. 8, No. 3, pp. 343-349, 1994 Copyright © 1994 ElsevierScienceLtd Printed in Great Britain. All rights reserved 0887-2333/94 $7.00 + 0.00
A D E N I N E A N D PYRIDINE NUCLEOTIDE CONCENTRATIONS A N D RELATIONSHIPS TO 2,6-DINITROTOLUENE METABOLISM IN CULTURED RAT LIVER SLICES* D. E. CHAPMANt,S. R. MICHENER~and G. POWlS§ i'Division of Pharmacokinetics and Drug Metabolism, Wellcome Research Laboratories, Burroughs Wellcome Co., Research Triangle Park, NC 27709, :~Department of Physiology, Mayo Clinic and Foundation, Rochester, Minnesota and §Arizona Cancer Center, University of Arizona, Tucson, Arizona, USA (Received 26 March 1993; revisions received 12 October 1993)
Abstract--The relationships between nucleotide (ATP, ADP, AMP, NADPH, NADP + , NADH and NAD +) concentrations and the metabolism of the hepatocarcinogen 2,6-dinitrotoluene (2,6-DNT) in cultured slices of rat liver were investigated. ATP, NADPH and NADH concentrations in freshly prepared rat liver slices at the beginning of culture were 48-67% lower than those measured in freeze-clamped rat liver (in vivo values). ATP concentrations in cultured liver slices increased with time of incubation, and after 4 hr ATP concentrations in liver slices were 25% greater than in vivo values. In contrast, NADPH and NADH concentrations did not recover with time of culture, and after 4 hr NADPH and NADH concentrations in liver slices were 50 and 24% of in vivo values, respectively. The addition of ethanol (50 mM) to cultured liver slices increased NADH concentrations by 132%, relative to untreated liver slices. Treatment of liver slices with ammonium chloride (10mM), 6-aminonicotinamide (1 mM), or the substitution of fructose for glucose in the incubation medium significantly decreased NADPH concentrations by 64, 44 and 63%, respectively. All treatments significantly decreased ATP concentrations; fructose was the most effective agent tested and decreased liver slice ATP concentrations by 69%. These results indicate that the changes in liver slice nucleotide concentrations that occur during culture are not irreversible, and we suggest that these changes are a homoeostatic response to culture conditions and not a reflection of tissue damage or cytotoxicity. Rates of 2,6-dinitrotoluene metabolism by rat liver slices were unaffected by fructose or ammonium chloride, despite the decrease in NADPH concentrations produced by these agents. These results suggest that NADPH concentrations are not rate-limiting to 2,6-DNT metabolism by rat liver slices.
INTRODUCTION Studies to compare the hepatic metabolism of xenobiotics in humans and laboratory animals have used a variety of systems, including subcellular fractions (Chapman et al., 1992; Seddon et al., 1987), isolated hepatocytes (Chenery et al., 1987; Fabre et al., 1990; Green et al., 1984) and liver slices (Chapman et al., 1990; Fisher et al., 1991). The development of the liver slice technique is particularly important for studies of xenobiotic metabolism in humans, because of the difficulties associated with the isolation of viable hepatocytes from small pieces of surgical waste tissue, whole organ perfusion, or in vivo studies with potentially hazardous xenobiotics (Powis et al., 1989). The factors that regulate xenobiotic
*Reports of part of this work were presented at the Society of Toxicology 1992 meeting in Seattle, Washington, USA. Abbreviations: 2,6-DNT = 2,6-dinitrotoluene; DMEM = Dulbecco's modified Eagle's medium. rtv 8/r-c
metabolism in cultured liver slices have not been well defined. Previous studies with rat liver slices indicate that oxygen, at ambient concentrations between 25 and 100%, is not rate-limiting to the metabolism of 2,6-dinitrotoluene (2,6-DNT) by the hepatic mixed-function oxidase system (Chapman et al., 1993). The microsomal mixed-function oxidase system plays a prominent role in the hepatic metabolism of a wide variety of xenobiotics. Factors that regulate in vitro rates of hepatic mixed-function oxidation reactions may include enzyme concentration (i.e. cytochrome P-450) or cofactor supply (i.e. N A D P H ) ; in most in vitro systems rates of mixed-function oxidase reactions are not usually limited by substrate or oxygen supply (reviewed in Thurman and Kauffman, 1980 and Thurman et al., 1987). In the present study we measured adenine and pyridine nucleotide concentrations in 0.3-mm-thick cultured rat liver slices prepared with a commercially available microtome (Brendel et al., 1987; Krumdieck et al., 1980) and compared these values with liver nucleotide concentrations measured in vivo. In addition, we
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examined the possible rate-limiting effects of NADPH concentrations on the metabolism of 2,6DNT, a substrate of the hepatic mixed-function oxidase system and an experimental hepatocarcinogen with potential widespread human exposure (Chapman et al., 1992; Leonard et al., 1987). MATERIALS AND METHODS
Chemicals. [3-3H]2,6-Dinitrotoluene (sp. act. 24Ci/mmol) in methanol was purchased from Moravek Biochemicals (Brea, CA, USA), and HPLC separation showed it to be more than 95% radiolabelled 2,6-DNT. The specific activity was adjusted to 20 mCi/mmol by addition of non-radioactive 2,6DNT to obtain a 2,6-DNT stock solution of 50 mM. This stock [3H]2,6-DNT was added at 2 pl/ml incubation medium to give a final 2,6-DNT concentration of 0.1 mM. The non-radioactive reference compounds 2,6diaminotoluene, 2-amino-6-nitrotoluene, 2,6-dinitrobenzaldehyde and 2,6-dinitrotoluene were obtained from Aldrich Chemical Co. (Milwaukee, WI, USA). 2,6-Dinitrobenzylalcohol and 2,6-dinitrobenzoic acid were synthesized from 2,6-dinitrobenzaldehyde, and the glucuronide conjugate of 2,6-dinitrobenzylalcohol was synthesized by rat liver microsomes as described previously (Chapman et al., 1993). Dulbecco's modified Eagle's medium without phenol red or bicarbonate (DMEM) was obtained from Gibco-BRL (Grand Island, NY, USA). All other reagents were from Sigma Chemical (St Louis, MO, USA). Tissue preparations. Rat liver was obtained from non-fasted male Fischer F344 rats, weighing 200-300g (Harlan Sprague-Dawley, Madison, WI, USA). Rat liver slices (approx. 0.3 mm thick) were prepared with a commercially available microtome (Brendel et al., 1987; Krumdieck et al., 1980) from cylindrical cores of liver tissue (8 mm in diameter) as described previously (Chapman et al., 1993). The liver cores and microtome blade were immersed in Krebs-Henseleit buffer (pH 7.4) during preparation of the slices. The liver slices were stored in DMEM (supplemented with 10% foetal calf serum and buffered to pH 7.4 with 25raM HEPES) for a maximum of 45 min before use. All solutions were maintained at 0-4°C and were saturated with 100% 02. Rates of 2,6-DNT metabolism and nucleotide concentrations were normalized to tissue wet weight, which was measured using slices that had been gently blotted dry and weighed before incubation. Freezeclamped male Fischer F344 rat liver was prepared, within 10-20 sec of decapitation of the animal, by rapidly compressing the liver in situ between two aluminium blocks chilled in liquid nitrogen. Incubations. Liver slices were incubated (t-2 slices/ml medium) in covered 35-mm petri dishes (Coming, Park Ridge, IL, USA) with sufficient agitation by an orbital shaker to maintain a constant
movement of the slice in the medium (Chapman et al., 1993). All incubations were conducted under 100% 02 at 37°C in DMEM (without foetal calf serum) buffered to pH 7.4 with 25 mM HEPES. For measurement of adenine and pyridine nucleotide concentrations, the liver slices were preincubated for 15 min, the incubation medium was replaced, and then the incubations were continued for the time specified. The effects on nueleotide concentrations in the liver slices of 10 mM ammonium chloride, 50 m~a ethanol, I mM 6-aminonicotinamide (all concentrations are final), or the substitution of fructose (4.5 g/litre) for glucose in the incubation medium were also determined. Ammonium chloride, ethanol, 6-aminonicotinamideor fructose were added after the 15-min preincubation described above, and the effects on nucleotide concentrations were determined after incubation for an additional 60 min. For the study of 2,6-DNT metabolism, liver slices were preincubated for 15 min, the incubation medium was changed, and the liver slices were treated for 60 min with 10 mM ammonium chloride or fructose (4.5 g/litre). 2,6-DNT metabolism was started by the addition of 0.1 mM 2,6-DNT and incubations were continued for an additional 60min. Controls were treated with an equal volume of methanol. Sample preparation. 2,6-DNT metabolism was stopped by transferring 2 ml incubation medium to 4 ml ethyl acetate-acetone (3 : 1, v/v) and immediately freezing the mixture at -40°C. Frozen samples were thawed and extracted as described previously (Chapman et al., 1992). The percentage recoveries of 2,6-DNT metabolites added to the incubation medium were estimated by UV absorbance to be between 77 and 100%, and rates of 2,6-DNT metabolism were adjusted for these percentage recoveries, In preliminary experiments, no formation of 2,6DNT metabolites by liver slices heated to 100% for 10 min could be detected. 2,6-DNT metabolites did not accumulate in liver slices and 2,6-DNT metabolites in liver slices represented 1-2% of the total metabolites produced (i.e. approximately equal to the liver slice volume). Pyridine and adenine nucleotide concentrations were determined in freeze-clamped rat liver and in rat liver slices frozen in liquid nitrogen at the time of sampling. Frozen tissues were immediately homogenized in a minimum of 10ml ice-cold chloroform-methanol (1:1, v/v) per g liver with a Potter-Elvehjem homogenizer and the homogenate was extracted three times with equal volumes of 67 mM Tris base (pH 11). The aqueous extracts were pooled, divided into two equal aliquots and sufficient 2 N HCI added to one aliquot to bring the pH to 4.5. Each aliquot was lyophilized, reconstituted in 0.25 ml distilled water and filtered through a 45-#m syringe filter (Gelman Sciences, Ann Arbor, MI, USA). The acidic fraction was assayed for ATP, ADP, AMP, NADP + and NAD + and the basic fraction was assayed for NADPH and NADH. All procedures
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Nucleotides and 2,6-DNT metabolism in liver slices Table I. Nucleotideconcentrationsin rat liverin viva and rat liverslicesin vitro
Nucleotide
Nucleotide conch in viva ( # m o l / g liver)
0 hr
0.5 hr
I hr
ATP ADP AMP NADP + NADPH NAD + NADH
2.78 + 0.20 1.19 +_ 0.02 0.300 _+ 0.024 0.083 + 0.010 0.321 _+0.028 0.844 _+ 0.055 0.089 +- 0.022
0.909 +- 0.182" 0.619 + 0.085* 0.170 __.0.114 0.130 + 0.019" 0.139.-_0.042" 0.349 + 0.028* 0.046 +- 0.021
1.36 + 0.23* 0.555 + 0.161" 0.140 + 0.076* 0.132 + 0.033* 0.153+0.040" 0.427 + 0.088* 0.029 _ 0.011"
1.74 __.0.74 0.452 + 0.146" 0.076 _+ 0.028* 0.152 __.0.036* 0.144.-,0.052" 0.477 __.0.211" 0.024 +- 0.01 I*
Nucleotide canon ( ~ m o l / g liver) in liver slices in vitro after incubation for§: 2 hr 2.58 0.402 0.082 0.184 0.171 0.567 0.021
+ 0.60t +_ 0.073"I" __.0.013* +- 0.017*~f .--0.033* + 0.065"$ + 0.008*
4 hr 3.50 + 1.04t 0.346 + 0.050*t 0. I 15 _ 0.053* 0.165 + 0.124 0.161 .--0.032* 0.636 + 0.241 0.021 + 0.017*
:~ln viva nucleotide concentrations were measured in male Fischer F344 rat liver which was freeze clamped within 20-30 sec of death. Values are means +- SD of three livers. §Liver slices were obtained from non-fasted male Fischer 344 rats. The slices were preincubated for 15 min, at which time (0 hr) the incubation medium was changed and the incubations were started. Incubations were carried out at 37°C under an atmosphere of 100% oxygen. Values are means _+ SD for four separate incubations at each time point. Values marked with an asterisk differ significantly from the value for the freeze-clamped (in viva) rat liver (*P < 0.05); values marked with a dagger differ significantly from the corresponding value for liver slices at 0 hr (~P < 0.05).
were performed at 0-4°C; samples were stored at 0°C for a maximum of 48 hr after extraction. There were minimal losses (2-5% in 24-48 hr at 0°C) of NADPH and NADH when stored in alkaline solution, or of NADP + , NAD + , ATP, ADP and AMP when stored in acid solution. Percentage recoveries of exogenous nucleotides added to rat liver homogenates were between 97 and 100%. H P L C analysis. 2,6-DNT and 2,6-DNT metabolites were separated by HPLC as described previously (Chapman et al., 1992). Radioactivity was measured by a radiochromatographic flow detector (Raytest-Ramona, IN/US Instruments, Fairfield, N J,
300 - -
200
--
2 100
Z 0
I
I
I
0
2
4
Time (hr) Fig. 1. Adenine and pyridine nucleotide ratios calculated from the data presented in Table 1. Nucleotide ratios for ATP: ADP (O), NAD + :NADH (I--1) and NADPH:NADP + (V) are normalized to ratios obtained in viva (Table 1). Nucleotide ratios in viva include 2.34 (ATP:ADP), 9.48 (NAD ÷:NADH) and 3.87 (NADPH:NADP ÷). Values are the means of four separate incubations at each time point and range bars indicate the SEM.
USA) and peak areas were converted to pmol product formed by reference to a standard curve prepared with [3H]2,6-DNT, with correction made for quenching differences in the solvent gradient steps. Nucleotides were separated by a modification of previous HPLC methods (Formato et al., 1990; Jones, 1981; Stocchi et al., 1985) on an Alltech nucleotide/nucleoside, 250 mm x 4.6 mm, 7-#m column (Alltech Associates, Deerfield, IL, USA) preceded by a Brownlee RP 18 guard column (Brownlee Laboratories, Santa Clara, CA, USA). For elution of the sample a 0.1 M KH2PO4 (pH 6)/methanol gradient system was used as follows: 12 min isocratic flow of KH2PO 4 buffer, a linear gradient to 90% buffer/10% methanol at 30 min, and an additional 10 min at 90% buffer/10% methanol. All flow rates were 1.0 ml/min. NADPH and NADH were detected by absorbance at 340nm; ATP, ADP, AMP, NADP ÷ and NAD ÷ were detected by absorbance at 254 nm. Nucleotide concentrations were calculated based on peak areas (measured at 254 nm) of an internal standard N6-(6-aminohexyl)adenosine 3',5'diphosphate (Sigma Chemical Co., St Louis, MO, USA) added to extracted samples. The detector response ratios of nucleotides to internal standard at equimolar concentrations were as follows: 0.74, ATP; 0.75, ADP; 0.70 AMP; 0.65, NADP+; 0.26, NADPH; 0.50, NAD+; 0.17, NADH. The detector response to nucleotide standards was linear from 1.0 nmol/ml to 1.3/~mol/ml at injection volumes of 100 pl. Limits of detection for nucleotides in rat liver were approximately 0.21 nmol/g liver (ATP, ADP, AMP, NADP ÷ and NAD ÷) and 0.96nmol/g liver (NADPH and NADH). Limits of detection were higher for reduced pyridine nucleotides because of their lower molar absorptivity at 340 nm in comparison with 254 nm. Statistical analysis. Statistically significant treatment effects at P < 0.05 were determined by analysis of variance, followed by Dunett's multiple comparison test to establish significance between controls and individual treatments at P <0.05 (Toothaker, 1991).
D. E. CHAPMAN et al.
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Nucleotide ATP ADP AMP NADP ÷ NADPH NAD ÷ NADH
Table 2. Effect of incubation conditions on nucleotide concentrations in rat liver slices Nucleotide Nucleotide conch (/amol/g liver) in cultures treated with: c o n c n in control cultures Ammonium 6-Amino(#mol/g liver) Fructose chloride Ethanol nicotinamide 1.89 + 0.33 0.584 + 0.080 0.103 + 0.050 0.161 ± 0.069 0.162 ± 0.057 0.513 ± 0.048 0.034 ± 0.011
0.584 + 0.028* 0.391 ± 0.029* 0.100 + 0.013 0.162 ± 0.020 0.060 ± 0.022* 0.419 ± 0.010" 0.018 ± 0.010
1.21 + 0.15" 0.571 + 0.061 0.199 + 0.026* 0.146 ± 0.012 0.058 ± 0.017" 0.509 ± 0.050 0.023 ± 0.004
1.15 + 0.28* 0,481 + 0.140 0.208 + 0.036* 0.065 ± 0.035 0.103 ± 0.019 0.467 ± 0.043 0.079 ± 0.030*
1.24 + 0.16" 0.609 ± 0.079 NDI" 0.275 ± 0.040* 0.090 ± 0.008 0.475 ± 0.060 0.034 ± 0.002
Liver slices from male Fischer F344 rats were preincubated for 15 rain, at which time the incubation medium was changed and treatments started. Liver slices were treated for 60 min at 37"C under an atmosphere of 100% oxygen. Treatments included substitution of fructose (4.5 g/litre) for glucose in the incubation medium, or addition of ammonium chloride (10 mM), ethanol (50 mM) or 6-am±non±cot±ham±de(I .0 mM). Values are means ± SD of four separate incubations, and those marked with an asterisk differ significantly from the control value (*P < 0.05). tAMP could not be quantitated in these samples because of co-elution with 6-aminonicotinamide.
RESULTS In vivo (i.e. f r e e z e - c l a m p e d rat liver) n u c l e o t i d e c o n c e n t r a t i o n s (Table 1) were similar to t h o s e t h a t have b e e n m e a s u r e d in p r e v i o u s studies using H P L C o r e n z y m a t i c m e t h o d s (Ferr6 et al., 1979; J o n e s , 1981; K a u f f m a n a n d Evans, 1977; K l i n g e n b e r g , 1985; W i l l i a m s o n a n d C o r k e y , 1969). In vivo n u c l e o t i d e ratios c a l c u l a t e d f r o m the values in Table 1 were 2.34 + 0.15 ( A T P : A D P ) , 3.87 _ 0.64 ( N A D P H : N A D P +), a n d 9.48 _+ 2.17 ( N A D + : N A D H ) . W i t h the e x c e p t i o n o f N A D H , N A D P + a n d A M P , initial n u c l e o t i d e c o n c e n t r a t i o n s in liver slices (0 hr) were significantly l o w e r t h a n in vivo values (Table 1). Initial n u c l e o t i d e ratios in liver slices [i.e. 1.47 _+ 0.43 ( A T P : A D P ) , 1.06 + 0.31 ( N A D P H : N A D P +) and 7.58 + 2.59 (NAD + : N A D H ) ] were also lower t h a n the ratios o b t a i n e d in vivo. T o t a l a d e n i n e nucleotide, N A D ( H ) a n d N A D P ( H ) c o n c e n t r a t i o n s in liver slices at the beginning o f i n c u b a t i o n (0 hr) were 40, 42 a n d 6 7 % o f in vivo values, respectively. A T P c o n c e n t r a t i o n s in liver slices increased with i n c u b a t i o n time, a n d after 4 h r liver slice A T P c o n c e n t r a t i o n s e x c e e d e d in vivo values (Table 1). Liver slice A T P : A D P ratios also i n c r e a s e d with time o f incub a t i o n , a n d w i t h i n 1 h r the ratio o f these n u c l e o t i d e s e x c e e d e d ratios m e a s u r e d in vivo (Fig. 1). R e d u c e d p y r i d i n e n u c l e o t i d e c o n c e n t r a t i o n s did n o t c h a n g e significantly d u r i n g i n c u b a t i o n , a n d N A D P H : N A D P + ratios r e m a i n e d at values similar to t h e initial liver slice ratio (1.06). H o w e v e r , N A D + c o n c e n t r a -
tions a n d N A D + : N A D H ratios i n c r e a s e d with time o f i n c u b a t i o n . W i t h i n 0.5 h r liver slice N A D ÷ : N A D H ratios e x c e e d e d in vivo values, a n d at 4 h r the N A D ÷ : N A D H ratio (30.2) in liver slices was a p p r o x i m a t e l y 2.3 times the ratio m e a s u r e d in vivo. T o t a l a d e n i n e nucleotide, N A D ( H ) a n d N A D P ( H ) c o n c e n t r a t i o n s r e c o v e r e d to 93, 70 a n d 8 1 % o f in vivo values after i n c u b a t i o n for 4 hr. Liver slice A T P c o n c e n t r a t i o n s (Table 2) a n d A T P : A D P ratios (Table 3) were significantly dec r e a s e d by a d d i t i o n o f a m m o n i u m chloride, e t h a n o l o r 6 - a m i n o n i c o t i n a m i d e , o r by s u b s t i t u t i o n o f f r u c t o s e for glucose in the i n c u b a t i o n media. F r u c t o s e was the m o s t effective a g e n t tested a n d p r o d u c e d a 6 9 % decrease in A T P c o n t e n t a n d a 59% decrease in A T P : A D P ratios. F r u c t o s e a n d a m m o n i u m c h l o r i d e significantly d e c r e a s e d liver slice N A D P H c o n t e n t s by 63 a n d 6 4 % (Table 2) a n d d e c r e a s e d N A D P H : N A D P ÷ ratios by 63 a n d 6 7 % (Table 3). T h e 4 4 % decrease in N A D P H c o n c e n t r a t i o n s p r o d u c e d by 6a m i n o n i c o t i n a m i d e (Table 2) was n o t significantly different f r o m the control; h o w e v e r , 6 - a m i n o n i c o t i n am±de significantly d e c r e a s e d N A D P H : N A D P ÷ ratios by 6 0 % in c u l t u r e d liver slices (Table 3). E t h a n o l significantly increased liver slice N A D H c o n t e n t s by 132% (Table 2) a n d d e c r e a s e d N A D + : N A D H ratios to 3 9 % o f c o n t r o l values (Table 3). 2 , 6 - D N T m e t a b o l i s m by liver slices was n o t significantly affected by a m m o n i u m c h l o r i d e o r the substit u t i o n o f f r u c t o s e for glucose in the i n c u b a t i o n m e d i u m (Table 4). R a t e s o f 2 , 6 - D N T m e t a b o l i s m by
Table 3. Effect of incubation conditions on nucleotide ratios in rat liver slices Treatment ATP: ADP NADPH:NADP + NAD + : NADH Control 3.2 + 0.4 1.0 + 0.4 15.1 -F 5.2 Fructose 1.5+0.1" 0.37+0.11" 23.3+ 13.1 Ammonium chloride 2. I ± 0.2* 0.40 _.+0.10" 22.1 + 5.0 Ethanol 2.4 + 0.2* 1.6 ± 1.0 5.9 + 1.7* 6-Aminonicotinamide 2.0 + 0.2* 0.33 + 0.09* 14.0 ± 2.0 Nucleotide ratios were derived from the data presented in Table 2. Treatments included substitution of fructose (4.5 g/litre) for glucose in the incubation medium, or addition of ammonium chloride (10mM), ethanol (50mM), or 6-aminonicotinamide (1.0 mM). Values are the means + SD of four separate incubations, and those marked with an asterisk differ significantly from the control value (*P < 0.05).
Nucleotides and 2,6-DNT metabolism in liver slices Table 4. Effect of modifiers of nucleotide concentrations on the metabolism of 2,6-dinitrotoluene (2,6-DNT) by rat liver slices
Oxidized metabolites Modifier
(nmol/min/g liver)*
1.90 +__0.32 1.46 + 0.35 Fructose 2.01 + 0,45 Rat liver sliceswere preincubated for 15 rain, the incubation medium was changed and ammonium chloride (I0 raM) was added or fructose (4.5 g/litre) was substituted for glucose in the incubation medium. After treatment with ammonium chloride or fructose for 60 rain, 2,6-DNT metabolism was started by the addition of 2#1 2,6DNT/ml incubation medium and incubations were conducted for an additional 60 rain. All incubations were conducted at 37°C under an atmosphere of 100% oxygen. Initial 2,6-DNT concentrations were 0.1 mM; controls were treated with equal volumes of methanol. *Oxidizedmetabolitesincluded 2,6-dinitrobenzylalcoholand minor amounts (7% of the total 2,6-DNT metabolites formed) of the 2,6-dinitrobenzylalcohol glucuronide. Values are the means + SD of four separate incubations. None (control)
Ammonium chloride
rat liver slices in these studies (1.90 nmol/min/g liver) were comparable with rates of metabolism obtained in previous studies (Chapman et al., 1993). As in previous studies, 2,6-dinitrobenzylalcohol was the primary metabolite produced by rat liver slices (Chapman et al., 1993); minor amounts of the glucuronide conjugate of 2,6-dinitrobenzylalcohol (less than 7% of total 2,6-DNT metabolism) and 2-amino-6-nitrotoluene (less than 10% of total 2,6D N T metabolism) were also formed (results not shown). DISCUSSION The present studies suggest that nucleotide concentrations, and ratios of oxidized to reduced pyridine nucleotides, in cultured rat liver slices differ significantly from values measured in vivo (i.e. freeze-clamped rat liver). In particular, concentrations of N A D H and N A D P H were decreased, N A D + :N A D H ratios were increased and N A D P H : N A D P + ratios, were decreased in comparison with in vivo values. Similar increases in N A D + : N A D H ratios (approximately two-fold after 4 hr) have been observed in rat hepatocytes during culture (Stubberfield and Cohen, 1988). In contrast, liver slice A T P concentrations recovered during culture and approached in vivo values within 2 hr of incubation. These results are also similar to those of previous studies in which it has been reported that the A T P contents of rat liver slices recover to preincubation levels within 4--8 hr of culture (Goethals et al., 1990; Sipes et al., 1987). The changes in liver nucleotide concentrations that occur in liver slices during preparation and culture do not appear to be irreversible, since nucleotide concentrations could be modulated by ethanol, 6aminonicotinamide, fructose and a m m o n i u m chloride. Thus, the seemingly aberrant N A D P H : N A D P + and N A D + : N A D H ratios observed in liver slices, relative to values measured in vivo, may be homoeo-
347
static responses to culture conditions and not a r e f l e c t i o n of tissue damage or cytotoxicity. The ability of cultured liver slices to maintain energy levels (ATP concentrations and A T P : A D P ratios) at or above in vivo levels tends to confirm this conclusion. In addition, previous results indicate that liver slices can be maintained under culture conditions similar to those used in the present studies for 10hr with minimal histologically identifiable tissue damage or leakage of cellular contents (Chapman et al., 1993). The extractive procedures used in the present studies provide estimates of total (free and bound) nucleotide concentrations and, therefore, may not accurately reflect the true redox state of the liver slice (i.e. the ratios of free N A D P H : N A D P + and free N A D + : N A D H ; Williamson et al., 1967). However, nucleotide concentrations and the ratios of oxidized to reduced pyridine nucleotides in cultured liver slices were sensitive to manipulation by a variety of treatments, and the effects of these treatments were similar to effects produced in other in vitro systems. For example, fructose also decreases A T P concentrations in hepatocytes and lowers A T P : A D P ratios in the perfused rat liver (Kauffman et al., 1979; Silva et al., 1991), ethanol increases N A D H concentrations in the isolated perfused rat liver (Reinke et al., 1979), a m m o n i u m chloride decreases free N A D P H : N A D P + ratios in isolated hepatocytes (Sies et al., 1977) and 6-aminonicotinamide decreases A T P : A D P ratios and inhibits N A D P H formation in the perfused rat liver (Belinsky et al., 1985; Kauffman et al., 1979). Previous studies have suggested that N A D P H concentrations in rat liver slices are sufficient to support menadione metabolism and limit menadione toxicity (Wright and Paine, 1992). Although the present studies suggest that N A D P H concentrations and ratios of N A D P H : N A D P + are decreased in cultured liver slices, there was no indication that N A D P H was rate-limiting to 2,6-DNT metabolism by rat liver slices. Thus, fructose and a m m o n i u m chloride had no effect on rates of 2,6-DNT metabolism despite significant decreases in liver slice N A D P H concentrations and N A D P H : N A D P + ratios produced by these treatments. The inability of fructose or a m m o n i u m chloride to affect 2,6-DNT metabolism was unexpected, since N A D P H concentrations appear to limit rates of mixed-function oxidase activities in other systems (Thurman et al., 1987). It is possible that rates of 2,6-DNT metabolism by rat liver may be so low that even minimal concentrations of N A D P H are sufficient to maintain maximal rates of 2,6-DNT metabolism. In the perfused mouse liver, a m m o n i u m chloride significantly decreases both N A D P H concentrations and p-nitroanisole metabolism (Wu et al., 1990); however, rates of p-nitroanisole metabolism by the perfused mouse liver (0.4 # m o l / m i n / g liver) are approximately 200-fold greater than rates of 2,6-DNT metabolism (1.90 nmol/min/g liver) by rat liver slices. In addition,
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D.E. CHAPMANet al.
although the rate-limiting effects of N A D P H on hepatic mixed-function oxidase activities have been established for fasted animals, the role of N A D P H in the control of mixed-function oxidase activities in fed animals is less clear (Thurman et al., 1987). In livers from fed animals, as were used in the studies reported here, several sources of N A D P H are available (Reinke et al., 1983) and rates of N A D P H formation may be sufficient to support maximal rates of 2,6D N T metabolism in spite of the significant decreases in N A D P H concentrations produced by fructose and a m m o n i u m chloride. In summary, the present results suggest that significant changes in rat liver nucleotide concentrations occur during the culture of liver slices. These changes may result from the homoeostatic responses of the liver slice to culture conditions rather than to tissue damage or cytotoxicity, since liver slice energy levels (i.e. A T P concentrations and A T P : A D P ratios) reached or exceeded in vivo values during tissue culture. O f particular interest is a significant decrease in N A D P H concentrations and N A D P H : N A D P ÷ ratios in liver slices during culture, since previous studies suggest that N A D P H concentrations may limit mixed-function oxidase activities in the liver (Thurman et al., 1987). However, decreased liver slice N A D P H concentrations were not accompanied by a decrease in 2,6-DNT metabolism and this suggests that rates of 2,6-DNT mixed-function oxidase metabolism are not limited by the supply of reducing equivalents in cultured rat liver slices. Additional studies are required to characterize further the factors that affect rates of xenobiotic metabolism in cultured liver slices. It will be important to characterize the factors that control rates of xenobiotic metabolism in liver slices from fasted animals, to use alternative mixed-function oxidase substrates with higher rates of metabolism, and to determine whether increased liver enzyme (i.e. cytochrome P-450) content affects the rates of metabolism of xenobiotics by the liver slices. The results of such studies can then be applied to the in vitro characterization of xenobiotic metabolism by slices of human liver. Acknowledgements--This work was supported by grant ES-05304. D.E.C. was supported by the Society of Toxicology/Colgate-Palmolive Post-doctoral Fellowship Award in In Vitro Toxicology.
REFERENCES Belinsky S. A., Reinke L. A., Scholz R., Kauffman F. C. and Thurman R. G. (1985) Rates of pentose cycle flux in perfused rat liver: evaluation of the role of reducing equivalents from the pentose cycle for mixed-function oxidation. Molecular Pharmacology 78, 371-376. Brendel K., Gandolfi A. J., Krumdieck C. L. and Smith P. F. (1987) Tissue slicing and culturing revisited. Trends in Pharmacological Sciences 8, 11-15. Chapman D. E., Michener S. R. and Powis G. (1992) Metabolism of [3H]2,6-dinitrotoluene by human and rat liver microsomal and cytosolic fractions. Xenobiotica 22, 1015-1028.
Chapman D. E., Michener S. R. and Powis G. (1993) In vitro metabolism of [aH]2,6-dinitrotoluene by human
and rat liver. Toxicology in Vitro 3, 213-220. Chapman D. E., Moore T. J., Michener S. R., and Powis G.
(1990)
Metabolism
and
covalent
binding
of
[14C]toluene by human and rat liver microsomal fractions and liver slices. Drug Metabolism and Disposition 18, 929-936. Chenery R. J., Ayrton A., Oldham H. G., Standring P., Norman S. J., Seddon T. and Kirby R. (1987) Diazepam metabolism in cultured hepatocytes from rat, rabbit, dog, guiea pig, and man. Drug Metabolism and Disposition 15, 312-317. Fabre G., Combalbert J., Berger Y. and Cano J. P. (1990) Human hepatocytes as a key in vitro model to improve preclinical drug development. European Journal of Drug Metabolism and Pharmacokinetics 15, 165-171. Ferre P., Pegorier J.-P., Williamson D. H. and Girard J. (1979) Interactions in vivo between oxidation of non-esterified fatty acids and gluconeogenesis in the newborn rat. Biochemical Journal 182, 593-598. Fisher R., Barr J., Zukoski C. F., Putnam C. W., Sipes I. G., Gandolfi A. J. and Brendel K. (1991) In vitro hepatotoxicity of three dichlorobenzene isomers in human liver slices. Human and Experimental Toxicology 10, 357-363. Formato M., Masala B. and De Luca G. (1990) The levels of adenine nucleotides and pyridine coenzymes in red blood cells from the newborn, determined simultaneously by HPLC. Clinica Chimica Acta 189, 131 139. Goethals F., Deboyser D., Lefebvre V., De Coster I. and Roberfroid M. (1990) Adult rat liver slices as a model for studying the hepatotoxicity of vincaalkaloids. Toxicology in Vitro 4, 435-438. Green C. E., Dabbs J. E. and Tyson C. A. (1984) Metabolism and cytotoxicity of acetaminophen in hepatocytes isolated from resistant and susceptible species. Toxicology and Applied Pharmacology 76, 139-149. Jones D. P. (1981) Determination of pyridine dinucleotides in cell extracts by high-performance liquid chromatography. Journal of Chromatography 225, 446-449. Kauffman F. C. and Evans R. K. (1977) Alterations in nicotinamide and adenine nucleotide systems during mixed-function oxidation of p-nitroanisole in perfused livers from normal and phenobarbital-treated rats. Biochemical Journal 166, 583 592. Kauffman F. C., Evans R. K., Reinke L. A. and Thurman R. G. (1979) Regulation of p-nitroanisole Odemethylation in perfused rat liver: adenine nucleotide inhibition of NADP+-dependent dehydrogenases and NADPH-cytochrome c reductase. Biochemical Journal 184, 675~i81, Klingenberg M. (1985) Nicotinamide-adenine dinucleotides and dinucleotide phosphates (NAD, NADP, NADH, and NADPH). In Methods of Enzymatic Analysis. Vol. VII. Edited by H. U. Bergmeyer, Chapter 3.3, pp, 251 284. VCH Verlagsgesellschaft, Weinheim. Krumdieck C. L., dos Santos J. E. and Ho K.-J. (1980) A new instrument for the rapid preparation of tissue slices. Analytical Biochemistry 104, 118-123. Leonard T. B., Graichen M. E. and Popp J. A. (1987) Dinitrotoluene isomer-specific hepatocarcinogenesis in F344 rats. Journal of the National Cancer Institute 79, 1313-1319. Powis G., Melder D. C. and Wilke T. J. (1989) Human and dog, but not rat, isolated hepatocytes have decreased foreign compound-metabolizing activity compared to liver slices. Drug Metabolism and Disposition 17, 526-531. Reinke L. A., Kauffman F. C., Belinsky S. A. and Thurman R. G. (1983) Inhibition of p-nitroanisole O-demethylation in perfused rat liver by potassium cyanide. Archives of Biochemistry and Biophysics 225, 313-319.
Nucleotides and 2,6-DNT metabolism in liver slices Reinke L. A., Kauffman F. C. and Thurman R. G. (1979) Stimulation of p-nitroanisole O-demethylation by ethanol in perfused livers from fasted rats. Journal of Pharmacology and Experimental Therapeutics 211, 133-139. Seddon C. E., Boobis A. R. and Davies D. S. (1987) Comparative activation of paracetamol in the rat, mouse and man. Archives of Toxicology 11 (Suppl), 305-309. Sies H., Akerboom T. P. M. and Tager J. M. (1977) Mitochondrial and cytosolic NADPH systems and isocitrate dehydrogenase indicator metabolites during ureogenesis from ammonia in isolated rat hepatocytes. European Journal of Biochemistry 72, 301-307. Silva J. M., McGirr L. and O'Brien P. J. (1991) Prevention of nitrofurantoin-induced cytotoxicity in isolated hepatocytes by fructose. Archives of Biochemistry and Biophysics 289, 313-318. Sipes I. G., Fisher R. L., Smith P. F., Stine E. R., Gandolfi A. J. and Brendel K. (1987) A dynamic liver culture system: a tool for studying chemical biotransformation and toxicity. Archives of Toxicology 11, 20-23. Stocchi V., Cucchiarini L., Magnani M., Chiarantini L., Palma P. and Crescentini G. (1985) Simultaneous extraction and reverse-phase high-performance liquid chromatographic determination of adenine and pyridine nucleotides in human red blood cells. Analytical Biochemistry 146, 118-124.
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Stubberfield C. R. and Cohen G. M. (1988) NAD + depletion and cytotoxicity in isolated hepatocytes. Biochemical Pharmacology 37, 3967-3974. Thurman R. G., Belinsky S. A. and Kauffman F. C. (1987) Regulation of monooxygenation in intact cells. In Mammalian Cytochromes P-d50. Vol. II. Edited by F. P. Guengerich. pp. 131-152. CRC Press Inc., Boca Raton, FU Thurman R. G. and Kauffman F. C. (1980) Factors regulating drug metabolism in intact hepatocytes. Pharmacology Reviews 31, 229-251. Toothaker L. E. (1991) Multiple Comparisons for Researchers, pp. 59-61. Sage Publications, London. Williamson J. R. and Corkey B. E. (1969) Assays of intermediates of the citric acid cycle and related compounds by fluorometric methods. Methods in Enzymology 13, 434-513. Williamson D. H., Lund P. and Krebs H. A. (1967) The redox state of free nicotinamide-adenine dinucleotide in the cytoplasm and mitochondria of rat liver. Biochemical Journal 103, 514-527. Wright M. C. and Paine A. J. (1992) Resistance of precisioncut liver slices to the toxic effects of menadione. Toxicology in Vitro 6, 475-481. Wu Y.-R., Kauffman F. C., Qu W., Ganey P. and Thurman R. G. (1990) Unique role of oxygen in regulation of hepatic monooxygenation and glucuronidation. Molecular Pharmacology" 38, 128-133.
0887-2333(94)E0026-P
Toxic'. in Vitro Vol. 8, No. 3, pp. 343-349, 1994 Copyright © 1994 ElsevierScienceLtd Printed in Great Britain. All rights reserved 0887-2333/94 $7.00 + 0.00
A D E N I N E A N D PYRIDINE NUCLEOTIDE CONCENTRATIONS A N D RELATIONSHIPS TO 2,6-DINITROTOLUENE METABOLISM IN CULTURED RAT LIVER SLICES* D. E. CHAPMANt,S. R. MICHENER~and G. POWlS§ i'Division of Pharmacokinetics and Drug Metabolism, Wellcome Research Laboratories, Burroughs Wellcome Co., Research Triangle Park, NC 27709, :~Department of Physiology, Mayo Clinic and Foundation, Rochester, Minnesota and §Arizona Cancer Center, University of Arizona, Tucson, Arizona, USA (Received 26 March 1993; revisions received 12 October 1993)
Abstract--The relationships between nucleotide (ATP, ADP, AMP, NADPH, NADP + , NADH and NAD +) concentrations and the metabolism of the hepatocarcinogen 2,6-dinitrotoluene (2,6-DNT) in cultured slices of rat liver were investigated. ATP, NADPH and NADH concentrations in freshly prepared rat liver slices at the beginning of culture were 48-67% lower than those measured in freeze-clamped rat liver (in vivo values). ATP concentrations in cultured liver slices increased with time of incubation, and after 4 hr ATP concentrations in liver slices were 25% greater than in vivo values. In contrast, NADPH and NADH concentrations did not recover with time of culture, and after 4 hr NADPH and NADH concentrations in liver slices were 50 and 24% of in vivo values, respectively. The addition of ethanol (50 mM) to cultured liver slices increased NADH concentrations by 132%, relative to untreated liver slices. Treatment of liver slices with ammonium chloride (10mM), 6-aminonicotinamide (1 mM), or the substitution of fructose for glucose in the incubation medium significantly decreased NADPH concentrations by 64, 44 and 63%, respectively. All treatments significantly decreased ATP concentrations; fructose was the most effective agent tested and decreased liver slice ATP concentrations by 69%. These results indicate that the changes in liver slice nucleotide concentrations that occur during culture are not irreversible, and we suggest that these changes are a homoeostatic response to culture conditions and not a reflection of tissue damage or cytotoxicity. Rates of 2,6-dinitrotoluene metabolism by rat liver slices were unaffected by fructose or ammonium chloride, despite the decrease in NADPH concentrations produced by these agents. These results suggest that NADPH concentrations are not rate-limiting to 2,6-DNT metabolism by rat liver slices.
INTRODUCTION Studies to compare the hepatic metabolism of xenobiotics in humans and laboratory animals have used a variety of systems, including subcellular fractions (Chapman et al., 1992; Seddon et al., 1987), isolated hepatocytes (Chenery et al., 1987; Fabre et al., 1990; Green et al., 1984) and liver slices (Chapman et al., 1990; Fisher et al., 1991). The development of the liver slice technique is particularly important for studies of xenobiotic metabolism in humans, because of the difficulties associated with the isolation of viable hepatocytes from small pieces of surgical waste tissue, whole organ perfusion, or in vivo studies with potentially hazardous xenobiotics (Powis et al., 1989). The factors that regulate xenobiotic
*Reports of part of this work were presented at the Society of Toxicology 1992 meeting in Seattle, Washington, USA. Abbreviations: 2,6-DNT = 2,6-dinitrotoluene; DMEM = Dulbecco's modified Eagle's medium. rtv 8/r-c
metabolism in cultured liver slices have not been well defined. Previous studies with rat liver slices indicate that oxygen, at ambient concentrations between 25 and 100%, is not rate-limiting to the metabolism of 2,6-dinitrotoluene (2,6-DNT) by the hepatic mixed-function oxidase system (Chapman et al., 1993). The microsomal mixed-function oxidase system plays a prominent role in the hepatic metabolism of a wide variety of xenobiotics. Factors that regulate in vitro rates of hepatic mixed-function oxidation reactions may include enzyme concentration (i.e. cytochrome P-450) or cofactor supply (i.e. N A D P H ) ; in most in vitro systems rates of mixed-function oxidase reactions are not usually limited by substrate or oxygen supply (reviewed in Thurman and Kauffman, 1980 and Thurman et al., 1987). In the present study we measured adenine and pyridine nucleotide concentrations in 0.3-mm-thick cultured rat liver slices prepared with a commercially available microtome (Brendel et al., 1987; Krumdieck et al., 1980) and compared these values with liver nucleotide concentrations measured in vivo. In addition, we
343
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D.E. CHAPMANet al.
examined the possible rate-limiting effects of NADPH concentrations on the metabolism of 2,6DNT, a substrate of the hepatic mixed-function oxidase system and an experimental hepatocarcinogen with potential widespread human exposure (Chapman et al., 1992; Leonard et al., 1987). MATERIALS AND METHODS
Chemicals. [3-3H]2,6-Dinitrotoluene (sp. act. 24Ci/mmol) in methanol was purchased from Moravek Biochemicals (Brea, CA, USA), and HPLC separation showed it to be more than 95% radiolabelled 2,6-DNT. The specific activity was adjusted to 20 mCi/mmol by addition of non-radioactive 2,6DNT to obtain a 2,6-DNT stock solution of 50 mM. This stock [3H]2,6-DNT was added at 2 pl/ml incubation medium to give a final 2,6-DNT concentration of 0.1 mM. The non-radioactive reference compounds 2,6diaminotoluene, 2-amino-6-nitrotoluene, 2,6-dinitrobenzaldehyde and 2,6-dinitrotoluene were obtained from Aldrich Chemical Co. (Milwaukee, WI, USA). 2,6-Dinitrobenzylalcohol and 2,6-dinitrobenzoic acid were synthesized from 2,6-dinitrobenzaldehyde, and the glucuronide conjugate of 2,6-dinitrobenzylalcohol was synthesized by rat liver microsomes as described previously (Chapman et al., 1993). Dulbecco's modified Eagle's medium without phenol red or bicarbonate (DMEM) was obtained from Gibco-BRL (Grand Island, NY, USA). All other reagents were from Sigma Chemical (St Louis, MO, USA). Tissue preparations. Rat liver was obtained from non-fasted male Fischer F344 rats, weighing 200-300g (Harlan Sprague-Dawley, Madison, WI, USA). Rat liver slices (approx. 0.3 mm thick) were prepared with a commercially available microtome (Brendel et al., 1987; Krumdieck et al., 1980) from cylindrical cores of liver tissue (8 mm in diameter) as described previously (Chapman et al., 1993). The liver cores and microtome blade were immersed in Krebs-Henseleit buffer (pH 7.4) during preparation of the slices. The liver slices were stored in DMEM (supplemented with 10% foetal calf serum and buffered to pH 7.4 with 25raM HEPES) for a maximum of 45 min before use. All solutions were maintained at 0-4°C and were saturated with 100% 02. Rates of 2,6-DNT metabolism and nucleotide concentrations were normalized to tissue wet weight, which was measured using slices that had been gently blotted dry and weighed before incubation. Freezeclamped male Fischer F344 rat liver was prepared, within 10-20 sec of decapitation of the animal, by rapidly compressing the liver in situ between two aluminium blocks chilled in liquid nitrogen. Incubations. Liver slices were incubated (t-2 slices/ml medium) in covered 35-mm petri dishes (Coming, Park Ridge, IL, USA) with sufficient agitation by an orbital shaker to maintain a constant
movement of the slice in the medium (Chapman et al., 1993). All incubations were conducted under 100% 02 at 37°C in DMEM (without foetal calf serum) buffered to pH 7.4 with 25 mM HEPES. For measurement of adenine and pyridine nucleotide concentrations, the liver slices were preincubated for 15 min, the incubation medium was replaced, and then the incubations were continued for the time specified. The effects on nueleotide concentrations in the liver slices of 10 mM ammonium chloride, 50 m~a ethanol, I mM 6-aminonicotinamide (all concentrations are final), or the substitution of fructose (4.5 g/litre) for glucose in the incubation medium were also determined. Ammonium chloride, ethanol, 6-aminonicotinamideor fructose were added after the 15-min preincubation described above, and the effects on nucleotide concentrations were determined after incubation for an additional 60 min. For the study of 2,6-DNT metabolism, liver slices were preincubated for 15 min, the incubation medium was changed, and the liver slices were treated for 60 min with 10 mM ammonium chloride or fructose (4.5 g/litre). 2,6-DNT metabolism was started by the addition of 0.1 mM 2,6-DNT and incubations were continued for an additional 60min. Controls were treated with an equal volume of methanol. Sample preparation. 2,6-DNT metabolism was stopped by transferring 2 ml incubation medium to 4 ml ethyl acetate-acetone (3 : 1, v/v) and immediately freezing the mixture at -40°C. Frozen samples were thawed and extracted as described previously (Chapman et al., 1992). The percentage recoveries of 2,6-DNT metabolites added to the incubation medium were estimated by UV absorbance to be between 77 and 100%, and rates of 2,6-DNT metabolism were adjusted for these percentage recoveries, In preliminary experiments, no formation of 2,6DNT metabolites by liver slices heated to 100% for 10 min could be detected. 2,6-DNT metabolites did not accumulate in liver slices and 2,6-DNT metabolites in liver slices represented 1-2% of the total metabolites produced (i.e. approximately equal to the liver slice volume). Pyridine and adenine nucleotide concentrations were determined in freeze-clamped rat liver and in rat liver slices frozen in liquid nitrogen at the time of sampling. Frozen tissues were immediately homogenized in a minimum of 10ml ice-cold chloroform-methanol (1:1, v/v) per g liver with a Potter-Elvehjem homogenizer and the homogenate was extracted three times with equal volumes of 67 mM Tris base (pH 11). The aqueous extracts were pooled, divided into two equal aliquots and sufficient 2 N HCI added to one aliquot to bring the pH to 4.5. Each aliquot was lyophilized, reconstituted in 0.25 ml distilled water and filtered through a 45-#m syringe filter (Gelman Sciences, Ann Arbor, MI, USA). The acidic fraction was assayed for ATP, ADP, AMP, NADP + and NAD + and the basic fraction was assayed for NADPH and NADH. All procedures
345
Nucleotides and 2,6-DNT metabolism in liver slices Table I. Nucleotideconcentrationsin rat liverin viva and rat liverslicesin vitro
Nucleotide
Nucleotide conch in viva ( # m o l / g liver)
0 hr
0.5 hr
I hr
ATP ADP AMP NADP + NADPH NAD + NADH
2.78 + 0.20 1.19 +_ 0.02 0.300 _+ 0.024 0.083 + 0.010 0.321 _+0.028 0.844 _+ 0.055 0.089 +- 0.022
0.909 +- 0.182" 0.619 + 0.085* 0.170 __.0.114 0.130 + 0.019" 0.139.-_0.042" 0.349 + 0.028* 0.046 +- 0.021
1.36 + 0.23* 0.555 + 0.161" 0.140 + 0.076* 0.132 + 0.033* 0.153+0.040" 0.427 + 0.088* 0.029 _ 0.011"
1.74 __.0.74 0.452 + 0.146" 0.076 _+ 0.028* 0.152 __.0.036* 0.144.-,0.052" 0.477 __.0.211" 0.024 +- 0.01 I*
Nucleotide canon ( ~ m o l / g liver) in liver slices in vitro after incubation for§: 2 hr 2.58 0.402 0.082 0.184 0.171 0.567 0.021
+ 0.60t +_ 0.073"I" __.0.013* +- 0.017*~f .--0.033* + 0.065"$ + 0.008*
4 hr 3.50 + 1.04t 0.346 + 0.050*t 0. I 15 _ 0.053* 0.165 + 0.124 0.161 .--0.032* 0.636 + 0.241 0.021 + 0.017*
:~ln viva nucleotide concentrations were measured in male Fischer F344 rat liver which was freeze clamped within 20-30 sec of death. Values are means +- SD of three livers. §Liver slices were obtained from non-fasted male Fischer 344 rats. The slices were preincubated for 15 min, at which time (0 hr) the incubation medium was changed and the incubations were started. Incubations were carried out at 37°C under an atmosphere of 100% oxygen. Values are means _+ SD for four separate incubations at each time point. Values marked with an asterisk differ significantly from the value for the freeze-clamped (in viva) rat liver (*P < 0.05); values marked with a dagger differ significantly from the corresponding value for liver slices at 0 hr (~P < 0.05).
were performed at 0-4°C; samples were stored at 0°C for a maximum of 48 hr after extraction. There were minimal losses (2-5% in 24-48 hr at 0°C) of NADPH and NADH when stored in alkaline solution, or of NADP + , NAD + , ATP, ADP and AMP when stored in acid solution. Percentage recoveries of exogenous nucleotides added to rat liver homogenates were between 97 and 100%. H P L C analysis. 2,6-DNT and 2,6-DNT metabolites were separated by HPLC as described previously (Chapman et al., 1992). Radioactivity was measured by a radiochromatographic flow detector (Raytest-Ramona, IN/US Instruments, Fairfield, N J,
300 - -
200
--
2 100
Z 0
I
I
I
0
2
4
Time (hr) Fig. 1. Adenine and pyridine nucleotide ratios calculated from the data presented in Table 1. Nucleotide ratios for ATP: ADP (O), NAD + :NADH (I--1) and NADPH:NADP + (V) are normalized to ratios obtained in viva (Table 1). Nucleotide ratios in viva include 2.34 (ATP:ADP), 9.48 (NAD ÷:NADH) and 3.87 (NADPH:NADP ÷). Values are the means of four separate incubations at each time point and range bars indicate the SEM.
USA) and peak areas were converted to pmol product formed by reference to a standard curve prepared with [3H]2,6-DNT, with correction made for quenching differences in the solvent gradient steps. Nucleotides were separated by a modification of previous HPLC methods (Formato et al., 1990; Jones, 1981; Stocchi et al., 1985) on an Alltech nucleotide/nucleoside, 250 mm x 4.6 mm, 7-#m column (Alltech Associates, Deerfield, IL, USA) preceded by a Brownlee RP 18 guard column (Brownlee Laboratories, Santa Clara, CA, USA). For elution of the sample a 0.1 M KH2PO4 (pH 6)/methanol gradient system was used as follows: 12 min isocratic flow of KH2PO 4 buffer, a linear gradient to 90% buffer/10% methanol at 30 min, and an additional 10 min at 90% buffer/10% methanol. All flow rates were 1.0 ml/min. NADPH and NADH were detected by absorbance at 340nm; ATP, ADP, AMP, NADP ÷ and NAD ÷ were detected by absorbance at 254 nm. Nucleotide concentrations were calculated based on peak areas (measured at 254 nm) of an internal standard N6-(6-aminohexyl)adenosine 3',5'diphosphate (Sigma Chemical Co., St Louis, MO, USA) added to extracted samples. The detector response ratios of nucleotides to internal standard at equimolar concentrations were as follows: 0.74, ATP; 0.75, ADP; 0.70 AMP; 0.65, NADP+; 0.26, NADPH; 0.50, NAD+; 0.17, NADH. The detector response to nucleotide standards was linear from 1.0 nmol/ml to 1.3/~mol/ml at injection volumes of 100 pl. Limits of detection for nucleotides in rat liver were approximately 0.21 nmol/g liver (ATP, ADP, AMP, NADP ÷ and NAD ÷) and 0.96nmol/g liver (NADPH and NADH). Limits of detection were higher for reduced pyridine nucleotides because of their lower molar absorptivity at 340 nm in comparison with 254 nm. Statistical analysis. Statistically significant treatment effects at P < 0.05 were determined by analysis of variance, followed by Dunett's multiple comparison test to establish significance between controls and individual treatments at P <0.05 (Toothaker, 1991).
D. E. CHAPMAN et al.
346
Nucleotide ATP ADP AMP NADP ÷ NADPH NAD ÷ NADH
Table 2. Effect of incubation conditions on nucleotide concentrations in rat liver slices Nucleotide Nucleotide conch (/amol/g liver) in cultures treated with: c o n c n in control cultures Ammonium 6-Amino(#mol/g liver) Fructose chloride Ethanol nicotinamide 1.89 + 0.33 0.584 + 0.080 0.103 + 0.050 0.161 ± 0.069 0.162 ± 0.057 0.513 ± 0.048 0.034 ± 0.011
0.584 + 0.028* 0.391 ± 0.029* 0.100 + 0.013 0.162 ± 0.020 0.060 ± 0.022* 0.419 ± 0.010" 0.018 ± 0.010
1.21 + 0.15" 0.571 + 0.061 0.199 + 0.026* 0.146 ± 0.012 0.058 ± 0.017" 0.509 ± 0.050 0.023 ± 0.004
1.15 + 0.28* 0,481 + 0.140 0.208 + 0.036* 0.065 ± 0.035 0.103 ± 0.019 0.467 ± 0.043 0.079 ± 0.030*
1.24 + 0.16" 0.609 ± 0.079 NDI" 0.275 ± 0.040* 0.090 ± 0.008 0.475 ± 0.060 0.034 ± 0.002
Liver slices from male Fischer F344 rats were preincubated for 15 rain, at which time the incubation medium was changed and treatments started. Liver slices were treated for 60 min at 37"C under an atmosphere of 100% oxygen. Treatments included substitution of fructose (4.5 g/litre) for glucose in the incubation medium, or addition of ammonium chloride (10 mM), ethanol (50 mM) or 6-am±non±cot±ham±de(I .0 mM). Values are means ± SD of four separate incubations, and those marked with an asterisk differ significantly from the control value (*P < 0.05). tAMP could not be quantitated in these samples because of co-elution with 6-aminonicotinamide.
RESULTS In vivo (i.e. f r e e z e - c l a m p e d rat liver) n u c l e o t i d e c o n c e n t r a t i o n s (Table 1) were similar to t h o s e t h a t have b e e n m e a s u r e d in p r e v i o u s studies using H P L C o r e n z y m a t i c m e t h o d s (Ferr6 et al., 1979; J o n e s , 1981; K a u f f m a n a n d Evans, 1977; K l i n g e n b e r g , 1985; W i l l i a m s o n a n d C o r k e y , 1969). In vivo n u c l e o t i d e ratios c a l c u l a t e d f r o m the values in Table 1 were 2.34 + 0.15 ( A T P : A D P ) , 3.87 _ 0.64 ( N A D P H : N A D P +), a n d 9.48 _+ 2.17 ( N A D + : N A D H ) . W i t h the e x c e p t i o n o f N A D H , N A D P + a n d A M P , initial n u c l e o t i d e c o n c e n t r a t i o n s in liver slices (0 hr) were significantly l o w e r t h a n in vivo values (Table 1). Initial n u c l e o t i d e ratios in liver slices [i.e. 1.47 _+ 0.43 ( A T P : A D P ) , 1.06 + 0.31 ( N A D P H : N A D P +) and 7.58 + 2.59 (NAD + : N A D H ) ] were also lower t h a n the ratios o b t a i n e d in vivo. T o t a l a d e n i n e nucleotide, N A D ( H ) a n d N A D P ( H ) c o n c e n t r a t i o n s in liver slices at the beginning o f i n c u b a t i o n (0 hr) were 40, 42 a n d 6 7 % o f in vivo values, respectively. A T P c o n c e n t r a t i o n s in liver slices increased with i n c u b a t i o n time, a n d after 4 h r liver slice A T P c o n c e n t r a t i o n s e x c e e d e d in vivo values (Table 1). Liver slice A T P : A D P ratios also i n c r e a s e d with time o f incub a t i o n , a n d w i t h i n 1 h r the ratio o f these n u c l e o t i d e s e x c e e d e d ratios m e a s u r e d in vivo (Fig. 1). R e d u c e d p y r i d i n e n u c l e o t i d e c o n c e n t r a t i o n s did n o t c h a n g e significantly d u r i n g i n c u b a t i o n , a n d N A D P H : N A D P + ratios r e m a i n e d at values similar to t h e initial liver slice ratio (1.06). H o w e v e r , N A D + c o n c e n t r a -
tions a n d N A D + : N A D H ratios i n c r e a s e d with time o f i n c u b a t i o n . W i t h i n 0.5 h r liver slice N A D ÷ : N A D H ratios e x c e e d e d in vivo values, a n d at 4 h r the N A D ÷ : N A D H ratio (30.2) in liver slices was a p p r o x i m a t e l y 2.3 times the ratio m e a s u r e d in vivo. T o t a l a d e n i n e nucleotide, N A D ( H ) a n d N A D P ( H ) c o n c e n t r a t i o n s r e c o v e r e d to 93, 70 a n d 8 1 % o f in vivo values after i n c u b a t i o n for 4 hr. Liver slice A T P c o n c e n t r a t i o n s (Table 2) a n d A T P : A D P ratios (Table 3) were significantly dec r e a s e d by a d d i t i o n o f a m m o n i u m chloride, e t h a n o l o r 6 - a m i n o n i c o t i n a m i d e , o r by s u b s t i t u t i o n o f f r u c t o s e for glucose in the i n c u b a t i o n media. F r u c t o s e was the m o s t effective a g e n t tested a n d p r o d u c e d a 6 9 % decrease in A T P c o n t e n t a n d a 59% decrease in A T P : A D P ratios. F r u c t o s e a n d a m m o n i u m c h l o r i d e significantly d e c r e a s e d liver slice N A D P H c o n t e n t s by 63 a n d 6 4 % (Table 2) a n d d e c r e a s e d N A D P H : N A D P ÷ ratios by 63 a n d 6 7 % (Table 3). T h e 4 4 % decrease in N A D P H c o n c e n t r a t i o n s p r o d u c e d by 6a m i n o n i c o t i n a m i d e (Table 2) was n o t significantly different f r o m the control; h o w e v e r , 6 - a m i n o n i c o t i n am±de significantly d e c r e a s e d N A D P H : N A D P ÷ ratios by 6 0 % in c u l t u r e d liver slices (Table 3). E t h a n o l significantly increased liver slice N A D H c o n t e n t s by 132% (Table 2) a n d d e c r e a s e d N A D + : N A D H ratios to 3 9 % o f c o n t r o l values (Table 3). 2 , 6 - D N T m e t a b o l i s m by liver slices was n o t significantly affected by a m m o n i u m c h l o r i d e o r the substit u t i o n o f f r u c t o s e for glucose in the i n c u b a t i o n m e d i u m (Table 4). R a t e s o f 2 , 6 - D N T m e t a b o l i s m by
Table 3. Effect of incubation conditions on nucleotide ratios in rat liver slices Treatment ATP: ADP NADPH:NADP + NAD + : NADH Control 3.2 + 0.4 1.0 + 0.4 15.1 -F 5.2 Fructose 1.5+0.1" 0.37+0.11" 23.3+ 13.1 Ammonium chloride 2. I ± 0.2* 0.40 _.+0.10" 22.1 + 5.0 Ethanol 2.4 + 0.2* 1.6 ± 1.0 5.9 + 1.7* 6-Aminonicotinamide 2.0 + 0.2* 0.33 + 0.09* 14.0 ± 2.0 Nucleotide ratios were derived from the data presented in Table 2. Treatments included substitution of fructose (4.5 g/litre) for glucose in the incubation medium, or addition of ammonium chloride (10mM), ethanol (50mM), or 6-aminonicotinamide (1.0 mM). Values are the means + SD of four separate incubations, and those marked with an asterisk differ significantly from the control value (*P < 0.05).
Nucleotides and 2,6-DNT metabolism in liver slices Table 4. Effect of modifiers of nucleotide concentrations on the metabolism of 2,6-dinitrotoluene (2,6-DNT) by rat liver slices
Oxidized metabolites Modifier
(nmol/min/g liver)*
1.90 +__0.32 1.46 + 0.35 Fructose 2.01 + 0,45 Rat liver sliceswere preincubated for 15 rain, the incubation medium was changed and ammonium chloride (I0 raM) was added or fructose (4.5 g/litre) was substituted for glucose in the incubation medium. After treatment with ammonium chloride or fructose for 60 rain, 2,6-DNT metabolism was started by the addition of 2#1 2,6DNT/ml incubation medium and incubations were conducted for an additional 60 rain. All incubations were conducted at 37°C under an atmosphere of 100% oxygen. Initial 2,6-DNT concentrations were 0.1 mM; controls were treated with equal volumes of methanol. *Oxidizedmetabolitesincluded 2,6-dinitrobenzylalcoholand minor amounts (7% of the total 2,6-DNT metabolites formed) of the 2,6-dinitrobenzylalcohol glucuronide. Values are the means + SD of four separate incubations. None (control)
Ammonium chloride
rat liver slices in these studies (1.90 nmol/min/g liver) were comparable with rates of metabolism obtained in previous studies (Chapman et al., 1993). As in previous studies, 2,6-dinitrobenzylalcohol was the primary metabolite produced by rat liver slices (Chapman et al., 1993); minor amounts of the glucuronide conjugate of 2,6-dinitrobenzylalcohol (less than 7% of total 2,6-DNT metabolism) and 2-amino-6-nitrotoluene (less than 10% of total 2,6D N T metabolism) were also formed (results not shown). DISCUSSION The present studies suggest that nucleotide concentrations, and ratios of oxidized to reduced pyridine nucleotides, in cultured rat liver slices differ significantly from values measured in vivo (i.e. freeze-clamped rat liver). In particular, concentrations of N A D H and N A D P H were decreased, N A D + :N A D H ratios were increased and N A D P H : N A D P + ratios, were decreased in comparison with in vivo values. Similar increases in N A D + : N A D H ratios (approximately two-fold after 4 hr) have been observed in rat hepatocytes during culture (Stubberfield and Cohen, 1988). In contrast, liver slice A T P concentrations recovered during culture and approached in vivo values within 2 hr of incubation. These results are also similar to those of previous studies in which it has been reported that the A T P contents of rat liver slices recover to preincubation levels within 4--8 hr of culture (Goethals et al., 1990; Sipes et al., 1987). The changes in liver nucleotide concentrations that occur in liver slices during preparation and culture do not appear to be irreversible, since nucleotide concentrations could be modulated by ethanol, 6aminonicotinamide, fructose and a m m o n i u m chloride. Thus, the seemingly aberrant N A D P H : N A D P + and N A D + : N A D H ratios observed in liver slices, relative to values measured in vivo, may be homoeo-
347
static responses to culture conditions and not a r e f l e c t i o n of tissue damage or cytotoxicity. The ability of cultured liver slices to maintain energy levels (ATP concentrations and A T P : A D P ratios) at or above in vivo levels tends to confirm this conclusion. In addition, previous results indicate that liver slices can be maintained under culture conditions similar to those used in the present studies for 10hr with minimal histologically identifiable tissue damage or leakage of cellular contents (Chapman et al., 1993). The extractive procedures used in the present studies provide estimates of total (free and bound) nucleotide concentrations and, therefore, may not accurately reflect the true redox state of the liver slice (i.e. the ratios of free N A D P H : N A D P + and free N A D + : N A D H ; Williamson et al., 1967). However, nucleotide concentrations and the ratios of oxidized to reduced pyridine nucleotides in cultured liver slices were sensitive to manipulation by a variety of treatments, and the effects of these treatments were similar to effects produced in other in vitro systems. For example, fructose also decreases A T P concentrations in hepatocytes and lowers A T P : A D P ratios in the perfused rat liver (Kauffman et al., 1979; Silva et al., 1991), ethanol increases N A D H concentrations in the isolated perfused rat liver (Reinke et al., 1979), a m m o n i u m chloride decreases free N A D P H : N A D P + ratios in isolated hepatocytes (Sies et al., 1977) and 6-aminonicotinamide decreases A T P : A D P ratios and inhibits N A D P H formation in the perfused rat liver (Belinsky et al., 1985; Kauffman et al., 1979). Previous studies have suggested that N A D P H concentrations in rat liver slices are sufficient to support menadione metabolism and limit menadione toxicity (Wright and Paine, 1992). Although the present studies suggest that N A D P H concentrations and ratios of N A D P H : N A D P + are decreased in cultured liver slices, there was no indication that N A D P H was rate-limiting to 2,6-DNT metabolism by rat liver slices. Thus, fructose and a m m o n i u m chloride had no effect on rates of 2,6-DNT metabolism despite significant decreases in liver slice N A D P H concentrations and N A D P H : N A D P + ratios produced by these treatments. The inability of fructose or a m m o n i u m chloride to affect 2,6-DNT metabolism was unexpected, since N A D P H concentrations appear to limit rates of mixed-function oxidase activities in other systems (Thurman et al., 1987). It is possible that rates of 2,6-DNT metabolism by rat liver may be so low that even minimal concentrations of N A D P H are sufficient to maintain maximal rates of 2,6-DNT metabolism. In the perfused mouse liver, a m m o n i u m chloride significantly decreases both N A D P H concentrations and p-nitroanisole metabolism (Wu et al., 1990); however, rates of p-nitroanisole metabolism by the perfused mouse liver (0.4 # m o l / m i n / g liver) are approximately 200-fold greater than rates of 2,6-DNT metabolism (1.90 nmol/min/g liver) by rat liver slices. In addition,
348
D.E. CHAPMANet al.
although the rate-limiting effects of N A D P H on hepatic mixed-function oxidase activities have been established for fasted animals, the role of N A D P H in the control of mixed-function oxidase activities in fed animals is less clear (Thurman et al., 1987). In livers from fed animals, as were used in the studies reported here, several sources of N A D P H are available (Reinke et al., 1983) and rates of N A D P H formation may be sufficient to support maximal rates of 2,6D N T metabolism in spite of the significant decreases in N A D P H concentrations produced by fructose and a m m o n i u m chloride. In summary, the present results suggest that significant changes in rat liver nucleotide concentrations occur during the culture of liver slices. These changes may result from the homoeostatic responses of the liver slice to culture conditions rather than to tissue damage or cytotoxicity, since liver slice energy levels (i.e. A T P concentrations and A T P : A D P ratios) reached or exceeded in vivo values during tissue culture. O f particular interest is a significant decrease in N A D P H concentrations and N A D P H : N A D P ÷ ratios in liver slices during culture, since previous studies suggest that N A D P H concentrations may limit mixed-function oxidase activities in the liver (Thurman et al., 1987). However, decreased liver slice N A D P H concentrations were not accompanied by a decrease in 2,6-DNT metabolism and this suggests that rates of 2,6-DNT mixed-function oxidase metabolism are not limited by the supply of reducing equivalents in cultured rat liver slices. Additional studies are required to characterize further the factors that affect rates of xenobiotic metabolism in cultured liver slices. It will be important to characterize the factors that control rates of xenobiotic metabolism in liver slices from fasted animals, to use alternative mixed-function oxidase substrates with higher rates of metabolism, and to determine whether increased liver enzyme (i.e. cytochrome P-450) content affects the rates of metabolism of xenobiotics by the liver slices. The results of such studies can then be applied to the in vitro characterization of xenobiotic metabolism by slices of human liver. Acknowledgements--This work was supported by grant ES-05304. D.E.C. was supported by the Society of Toxicology/Colgate-Palmolive Post-doctoral Fellowship Award in In Vitro Toxicology.
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