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31P-NMR spectroscopy of perifused rat hepatocytes immobilized in agarose threads: application to chemical-induced hepatotoxicity
Biochimica et Biophysica Acta, 1139 (1992) 105-114 © 1992 Elsevier Science Publishers B.V. All rights reserved 0925-4439/92/$05.00
105
BBADIS 61158
31p_NMR spectroscopy of perifused rat hepatocytes immobilized in agarose threads: application to chemical-induced hepatotoxicity Hassan Farghali a, Lorenzo Rossaro a, Judith S. Gava]er b, David H. Van Thiel ~', Susan R. Dowd c, D o n a l d S. Williams d and Chien Ho c,d " Departments of Surgery, Unicersity of Pittsburgh School of Medicine, Pittsburgh, PA (USA), t, Department of Medicine, Unirersity of Pittsburgh School of Medicine, Pittsburgh, PA (USA), ~'Department of Biological Sciences, Carnegie Mellon Unicersity, Pittsburgh, PA (USA), and ,I Pittsburgh NMR Center for Biomedical Research, Carnegie Mellon Unirersity, Pittsburgh, PA (USA) (Received 12 December 1991)
Key words: NMR, 31p_; Hepatocyte; Hepatotoxicity; Ethanol; Acetaminophen; Allyl alcohol; Fructose; Agarose thread
A system consisting of isolated rat hepatocytes immobilized in agarose threads continuously perifused with oxygenated Krebs-Henseleit (KH) solution has been found to maintain cell viability with excellent metabolic activity for more than 6 h. The hepatocytes were monitored by phosphorus-31 nuclear magnetic resonance (3~p_NMR) spectroscopy at 4.7 Tesla, by measurement of oxygen consumption and by the leakage of lactate dehydrogenase (LD) and alanine aminotransferase (ALT). The data obtained were comparable to those found for an isolated perfused whole liver in vitro. The effects of allyl alcohol (AA), ethanol, and 4-acetaminophenol lAP) were examined. A solution of 225 IzM AA perifused for 90 min caused the disappearance of the /3-phosphate resonance of adenosine triphosphate (ATP) in the 3]P_NMR spectra, a 7-fold increase in LD leakage and a 70% reduction in oxygen consumption. Ethanol (1.0 M) perifused for 90 min reduced the /3-ATP signal intensity ratio by 20%, the phosphomonoester (PME) signal by 50% and inorganic phosphate (Pi) by 33% (P < 0.05). AP (10 mM) caused only mild liver-cell damage. The results demonstrate that perifused immobilized hepatocytes can be used as a liver model to assess the effects of a wide range of chemicals and other xenobiotics by NMR spectroscopy.
Introduction The application of nuclear magnetic resonance (NMR) spectroscopy to investigate metabolic parameters affecting hepatic function under different pathophysiological conditions has been documented during the past 11 years [1-6]. Few N M R spectroscopic studies, however, have been carried out on isolated liver cells [7]. N M R spectroscopic studies of isolated liver cells offer the advantage of precise control over the sample and the accuracy of quantitative measurements which can be carried out on a known type and number of ceils [8]. This is particularly important when studying
Abbreviations: KH, Krebs-Henseleit; 31p-NMR, phosphorus-31 nuclear magnetic resonance; LD, lactate dehydrogenase; ALT, alanine aminotransferase; AP, 4-acetaminophenol; ATP, adenosine triphosphate; AA, allyl alcohol; PME, phosphomonoester; Pi, inorganic phosphate. Correspondence: C. Ho, Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA, 15213-2683, USA.
the physiology and toxicology of cells non-invasively. To acquire meaningful information, an efficient perifusion bioreactor model needs to be used which allows for the provision of nutrients to the cells with and without drugs, the removal of cellular waste products, adequate oxygenation and the prevention of cell injury. In addition, a critical requirement for N M R studies is a relatively densely-packed cell suspension to obtain a spectrum with a reasonable signal-to-noise ratio and spectral resolution. Gillies et al. [8] have described various methods of cell immobilization and their application to cell biology including studies utilizing NMR. It is well recognized that immobilized cells have a greater stability than do non-immobilized cells [9]. The metabolite patterns of immobilized Chinese hamster lung fibroblasts have been monitored over many hours using 3~p-NMR [10,11] as well as pooled human lymphocytes [12] using N M R and 13C-labeled metabolites. 3~p-NMR studies have been applied to perifused interleukin-2-activated lymphocytes and tumor-infiltrating lymphocytes [13]. In these studies, it has been possible to separate intra-
106
cellular and extracellular information from the NMR spectra obtained for the perifused cells [14]. The method herein reported involves the application of real-time 3~p-NMR spectroscopy to the study of isolated hepatocytes immobilized in low-gelling agarose threads [10,15]. The metabolic behavior of isolated hepatocytes maintained in agarose gel was assessed under basal conditions and was compared to that obtained using a perfused whole liver. The response to allyl alcohol (AA), ethanol and 4-acetaminophenol (AP) perifusion at various concentrations and durations of exposure was also investigated. The compounds were chosen for their known ability to produce hepatic injuries which range from mild to severe [16-20]. Due to the medical and socioeconomic importance of alcohol, ethanol was chosen to be studied in the greatest detail. Additional conventional biochemical procedures were performed whenever possible. Materials and Methods
Sea plaque agarose (low-gelling t e m p e r a t u r e agarose) was obtained from FMC (Rockland, ME). Ethylene glycol-bis (/3-aminoethylether) N,N,N',N'-tetraacetic acid (EGTA), 4-acetaminophenol (AP) (acetaminophen), trypan blue, bovine serum albumin (BSA), methylenediphosphonic acid (MDPA) and diagnostic kits for the measurement of lactate dehydrogenase (LD) and adenosine-5'-triphosphate (ATP) were obtained from Sigma (St. Louis, MO). Collagenase was obtained from SEVAC (Institute Sera and Vaccines Prague). Allyl alcohol (AA) was obtained from Aldrich Chemical Company (Milwaukee, WI). Ethyl alcohol (ethanol), fructose and all other chemicals were reagent grade and were obtained from standard sources. Thin-
OUTSIDE THE MAGNET
Reservoir
wall Chem fluor T F E tubing (I.D. ().(12(~ in) was o b tained from B e r g h o f / A m e r i c a (Concord, (?A),
Preparation of the isolated hepatocytes Isolated hepatocytes were prepared using a twophase perfusion technique with the collagenase (0.67 m g / m l ) being added in the second phase as reported earlier [21,22]. After isolating and counting the recovered liver cells, viability was assessed both biochemically by LD leakage and histologically by trypan blue exclusion. Only cell preparations that excluded trypan blue to the extent of more than 95% were used. Thc cell density in all experiments was 4-5 - 1(17 cells/ml.
Immobilization of hepatocytes in agarose threads The 1.8% low-gelling agarose solution was prepared in warm Krebs-Henseleit (KH) solution (NaC1 6.99 g, KC1 0.36 g, K H 2 P O 4 0.13 g, MgSO 4- 7 H~O 0.295 g, C a C 1 2 . H 2 0 0.37, NaHCO 3 2 g per liter, pH 7.4) at 70°C. The agarose solution was brought to 37°C. The thermostated agarose solution was mixed 1:1 with hepatocytes which were isolated from fed SpragueDawley male rats, at a density of 4 - 5 . 107 cells/ml. The cells were immobilized in agarose threads by extruding the agarose-cell mixture through cooled Chem fluor T F E tubing into a 3-cm outer diameter NMR tube containing KH solution under carbogen (95% 0 2 and 5% CO:). The specific details of this technique have been described previously by Foxall et al. [10]. The threads were compressed gently to a final vol. of 20 ml which was entirely covered by the NMR coil. A solution of MDPA (100 retool, pH 9.5) sealed in a spherical glass bulb and positioned within the threads served as an external reference for both ~Lp chemical shifts and relative concentration determinations.
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Fig. 1. A schematic representation of the perifusion system of the hepatocytes immobilized in agarose threads. The left side represents a thermoregulated and oxygenated perifusion fluid flowing at 10 m l / m i n by p u m p 1. Pump 2 is connected to the perifusion system by a three-way stopcock to deliver the alcohol. The right side illustrates the interior of the magnet bore where a heat exchanger enables the inflow perifusate to be heated prior to flowing into the N M R tube.
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Perifusion of the threads: 31p-NMR spectroscopy, viability testing and biochemical analyses A schematic representation of the perifusion system for the hepatocytes immobilized in the agarose threads is shown in Fig. 1. The threads were perifused with a thermoregulated and well-oxygenated (95% 0 2 and 5% CO 2) K H medium at a rate of 10 m l / m i n in a non-recirculating system using a roller p u m p (pump 1). This perifusion rate was selected on the basis of its ability to maintain a high A T P level within the hepatocytes. In addition, it is known that under physiologic conditions, hepatic blood flow ranges from 100-130 m l / m i n per 100 g [23]. A second p u m p (pump 2) was used to deliver the compound of interest to the perifusate at a predetermined concentration using a threeway stopcock without affecting the final flow rate. Before initiating perifusion with the drug, the threads were equilibrated until a constant 3~P-NMR baseline spectrum and oxygen consumption rate were observed (about 60 min). These measurements were continued for a total of 6 h in the initial experiments to demonstrate that the hepatocytes immobilized in agarose threads were viable and stable for periods equal to those to be used in all subsequent experiments. Immobilized liver cell viability and function were assessed periodically by: (i) measuring 0 2 consumption calculated from rttPo2 , inflow outflow', blank -Po2 )-Po2 ]- flow rate; (ii) monitoring the percent of total LD leakage from cells at regular intervals for more than 6 h compared to the maximum lysate LD activity; (iii) measuring the fructose phosphorylation achieved after a fructose bolus; and (iv) measuring cytologically, in a separate experiment, the trypan blue (0.5%) exclusion after 6 h of perfusion. The A A used was diluted with K H solution to achieve a final concentration in the inflow perifusate of 100, 225 or 500/.tM. The AP was dissolved in ethanol and then diluted with K H solution to produce a final concentration of 2.5, 5 or 10 mM. In the AP experiments, the maximum ethanol concentration used to solubilize the AP in K H perifusate was 165 raM. Previous experiments have shown that this concentration of ethanol does not affect either the 31p-NMR spectra or the biochemical p a r a m e t e r s of the hepatocytes (results not shown). Ethanol was diluted with K H solution to provide a final concentration of 0.50, 0.85, 1.0 or 1.2 M in the perifusate. The actual concentrations obtained at initial exposure to the cells at the inflow and the outflow sites were determined at regular intervals as described in the Results section. Preliminary experiments showed that perifusion of an ethanol solution for 90 min followed by a wash-out with K H solution for 27 min gave highly reproducible results. The functional integrity of the cells was determined by measuring the oxygen consumption, LD and A L T leakage, as well as the fructose phosphorylation
response. The fructose studies consisted of perifusing the ceils with fructose in K H solution at a rate of 1.5 m m o l / m l per min delivered as a 1-min bolus. The area of the fructose-l-phosphate (F-l-P) signal in the 31p_ N M R spectrum was calculated for both control and ethanol perifused cells. In most cases, samples were obtained every 20 min from the outflow perifusate for various biochemical and oxygen consumption analyses, both during and after specific compound perifusion. Oxygen consumption was determined using an ABL2 acid-base laboratory instrument (Radiometer, Copenhagen). The results were calculated as n m o l / m i n per 106 hepatocytes. All other biochemical parameters such as LD leakage, /3-ATP, phosphomonoester (PME) and inorganic phosphate (Pi), were normalized to values achieved with 5 • 10 ~ cells. 3~p-NMR spectra were obtained at 81 MHz with a 3-cm solenoid coil at 37°C. Spectra were continuously recorded in blocks of 256 scans accumulated at a repetition rate of 2 s using a Bruker Biospec 1I spectrometer operating at 4.7 Tesla and equipped with a 40-cm bore-sized, horizontal superconducting solenoid. The areas under the peaks were determined using the integration routine available with the Bruker software. All signal areas reported are relative to the 31P-NMR reference compound, MDPA.
Functionality comparison with isolated perfused liver The abdomen of heparinized rats (500 U / r a t ) was entered via a mid[ine laparotomy and the infra-renal aorta was cannulated using a 20 G catheter. The liver was perfused in situ with 30 ml of K H solution after incising the inferior vena cava above and below the liver and clamping the aorta proximal to the celiac axis. The portal vein was cannulated using a second 20-G catheter whereby the liver was continuously perfused via the portal vein and the aorta with KH solution while the liver was being excised. The time from aortic clamping and initiation of the hepatic perifusion was less than 10 min in all cases. Perfusion of the liver was accomplished as described for thread perifusion. A bulb containing an external M D P A reference was included in the N M R sample tube.
Enzymatic evaluation and ATP in hepatocyte threads and whole liver Hepatocyte threads were prepared, perifused with K H solution for 1 h as described and then freeze clamped in liquid nitrogen. A portion of the frozen threads or about 1 g of livers obtained from rats under ether anaesthesia was ground in liquid nitrogen and 4% ( w / v ) pre-cooled perchloric acid (1:5, w / v ) and homogenized. The resultant homogenized samples were centrifuged at 4°C at 3000 rpm for 10 min. The supernatants were brought to p H 7 using 6 M K2CO 3 and
108 energy status for up to 6 h is shown in Fig. 2. In contrast, h e p a t o c y t c s isolated without t h r e a d i n g and p e r i f u s i o n s h o w e d no 3~P-NMR signals (results not shown). T h e viability o f p e r i f u s e d cells for p e r i o d s tip
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Fig. 2. ~IP-NMR spectra of immobilized perifused rat hepatocytes over a 6-h period at 37°C. I and It are spectra obtained after perifusion of 0 and 6 h, respectively. Peak assignments: (1) 13-phosphate of ATP; (2) oPphosphate of ATP; (3) y-phosphate of ATP; (4) inorganic phosphate (Pi); and (5) phosphomonoesters (PME). r e s p u n at 4°C at 3000 r p m a n d k e p t at - 7 0 ° C until assayed s p e c t r o p h o t o m e t r i c a l l y for t h e i r A T P c o n t e n t .
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Allyl alcohol induced hepatotoxicity in non-perifused isolated hepatocytes O n l y A A was t e s t e d using n o n - p e r i f u s e d i s o l a t e d h e p a t o c y t e s b e c a u s e it has t h e g r e a t e s t h e p a t o t o x i c i t y . H e p a t o c y t e s w e r e i n c u b a t e d for 20 rain at a c o n c e n t r a tion of 2 • 106 c e l l s / m i in a plastic t u b e at 37°C in K H solution at p H 7.4 u n d e r an a t m o s p h e r e of 95% O 2 a n d 5 % COz b e f o r e the a d d i t i o n o f A A at a c o n c e n t r a tion of 225 tzM. E x p e r i m e n t s using c o n t r o l u n t r e a t e d h e p a t o c y t e s w e r e run in parallel. Five r e p l i c a t e experim e n t s using d i f f e r e n t rats w e r e u s e d for e a c h t r e a t ment. A l i q u o t s w e r e o b t a i n e d at 0, 30, 60 a n d 120 rain after the A A a d d i t i o n for L D l e a k a g e m e a s u r e m e n t . E n z y m e l e a k a g e was m o n i t o r e d b o t h in an aliquot of cell-free m e d i u m a n d a f t e r lysis o f the cells with l % T r i t o n X-100. T h e l e a k a g e of e n z y m e u n d e r experim e n t a l c o n d i t i o n s was e x p r e s s e d as a p e r c e n t of the total lysate activity.
Statistical analysis A n a l y s i s of v a r i a n c e s with t h e T u k e y m u l t i p l e comp a r i s o n p r o c e d u r e was u s e d to e v a l u a t e c h a n g e s in r e c o r d e d v a r i a b l e s over time.
Results T h e ability o f h e p a t o c y t e s within the g e l - t h r e a d p e r ifusion system to r e a c h a n d m a i n t a i n a stable c e l l u l a r
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Fig. 3. (A) 31P-NMR spectra of immobilized rat hepatocytes at 37°C. After hepatocyte stabilization, fructose (F) was perifused at a rate of 1.5 mmol/ml/min in KH solution for 1 min. Spectra I - I l l were obtained I~efore, 14 and 32 min, respectively, after fructose perifusion. (B) 3~p-NMR spectra of perfused isolated rat liver at 37°C before (I) and 8 min after (ll) a fructose challenge. The immediate appearance of fructose-l-phosphate (peak 5) and its gradual disappearance over time is evident. The peak assignments are the same as in Fig. 2 (peak 6 is MDPA, the external reference).
109
to 6 h as assessed by trypan blue exclusion was 95%. The large Pi signal recorded in the 31P-NMR spectrum is due to the phosphate buffer as both the intra- and extracellular Pi contribute to the recorded Pi signal. The administration of a fructose load resulted in a sharp reduction in the Pi signal, suggesting that the P~ peak is dominated by intracellular Pi, a concomitant reduction in the ATP signal and a significant increase in the PME signal due to the accumulation of F-1-P as shown in Fig. 3A. Following the administration of a fructose challenge to an isolated perfused liver (Fig. 3B), a similar increase in the PME area was observed immediately with an associated reduction in the Pi and
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Fig. 5. Time-course of 225 /xM A A on ( A ) L D leakage and (B) the oxygen consumption of perifused immobilized hepatocytes in agarose threads. Time 0 represents the start of A A perifusion. (mean + S.E., I
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Fig. 4. 31p-NMR spectra of immobilized rat hepatocytes perifused with 225 g M A A at different time intervals at 37°C. Spectra I - V I are control, and 18, 50, 68, 80 a n d 90 min after A A perifusion, respectively.
n = 3).
ATP content of the liver. In several experiments with either KH-perifused cell gel threads or perfused whole liver, a 2-fold increase in PME due to F-1-P was seen. In both cases, 0 2 consumption and LD release were similar for several hours of perifusion. Hepatocytes in gel threads perifused with KH up to 6 h exhibited continued trypan blue exclusion (90%). The ATP content measured by enzymatic methods for perifused hepatocyte threads and whole liver was 45 nmol/106 cells and 3.8 ixmol/g, respectively, on average. Perifusion with A A produced measurable reductions in the phosphate pool at concentrations of 225 txM and above (results not shown). Fig. 4 shows the time-course for the 3~P-NMR spectra of the hepatocyte threads perifused with AA. A reduction in the ATP peaks was evident after 80 rain. A significant increase in the LD leakage and an induced reduction in 0 2 consumption were also seen (Fig. 5A and B). When isolated hepatocytes were incubated with 225 /xM AA, essentially
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Fig. 6. The time-course of 225 ~xM AA on the percent leakage of LD from isolated liver cell incubations. Time 0 represents the start of AA perifusion (mean + S . E . , n = 3 ) . * indicates significantly different (P < 0.05).
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100% LD leakage was obtained after 30 min of expos u r e (Fig. 6). A s e x p e c t e d , o n l y a v e r y f e w r e s i d u a l c e l l s w e r e a b l e t o e x c l u d e t r y p a n b l u e in t h e s e s t u d i e s . ethanol
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Fig. 7 3~p_NMR spectra of immobilized rat bepatocytes perifused at different ethanol concentrations at 37°C. I (control), II, II1 and IV are after perifusion with 0.85, 1.0 and 1.2 M ethanol, respectively. The peak assignments are the same as Fig. 2 (Peak 6 is the signal from the external standard MDPA).
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Fig. 8. The time-course of signal intensity ratios (mean _+S.E., n = 6): A, /3-ATP/MDPA; B, P M E / M D P A ; and C, P i / M D P A under the influence of 1.0 M ethanol perifusion for 90 rain. After ethanol perifusion and a washout, fructose (F) was perifused as a 1,5 mmol/ml per min bolus for 1 rain. The effect of ethanol at various concentrations on the 3Ip-NMR spectra obtained from the immobilized h e p a t o c y t e s is s h o w n in Fig. 7. A f t e r o b t a i n i n g s t a b l e
111 baseline spectra, the threads were perifused with 0.85, 1.0 or 1.2 M ethanol for 30 min followed by a 30-min washout period. These higher ethanol concentrations were selected because cells exposed to 0.50 M ethanol for 4 h did not show any change in their 31p-NMR (results not shown). From Fig. 7, a concentration-dependent decline in ATP and Pi with ethanol is evident• In order to study the detailed time-course of the effect of ethanol on cellular energy parameters, perifusion with 1.0 M ethanol was maintained for 90 min and the time-course of the /3-ATP, PME and Pi signal intensities were followed (Fig. 8A, B and C, respectively). Perifusion at this ethanol concentration resulted in a decline of about 20% in the /3-ATP level which plateaued and was statistically lower than the level during the subsequent perifusion period ( P < 0.05). After a 30-min washout with KH solution followed by a bolus of fructose, a further decline in t h e / 3 - A T P ratio occurred, followed by a slow recovery of the ATP signal to the pre-fructose level, but not to the preethanol basal level. The PME signals over these same six experiments showed a gradual decline with a minimum level achieved only at the end of the ethanolperifusion period. The administration of the fructose bolus was associated with a significant, sharp rise in the PME signal followed by a slower decrease to the prefructose level. The P~ signal followed the same pattern as the /3-ATP signal. Analyses of the perifusate for the cytosolic enzyme, LD, gave the results illustrated in Fig. 9. Ethanol perifusion produced a gradual but transient increase in
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Fig. 10. Time-course of oxygen consumption by immobilized hepatocytes in agarose threads (mean _+S.E., n = 6) before, during and after 1.0 M ethanol perifusion.
the level of enzyme in the perifusate for about 60 min, at which time the levels were significantly greater than those of the controls ( P < 0.05). Oxygen consumption was measured before, during, and after ethanol perifusion. The results are shown in Fig. 10. Under basal conditions, the immobilized cells consumed a mean of 21 nmol O 2 / m i n per 106 cells. Ethanol perifusion for 60 min reduced the 0 2 consumption significantly ( P < 0.05). After discontinuing the ethanol perifusion, O 2 consumption levels recovered slightly. The effect of different concentrations of AP on the 31p-NMR spectra obtained for the immobilized hepatocytes has been examined (results not shown). Only after 70 min of perifusion with 10 mM AP did a slight reduction occur in the /3-ATP peak resonance. This was accompanied by a slight increase in the LD leak and a slight decrease in the oxygen consumption at the highest concentration and duration of exposure.
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Fig. 9. Time-course of LD leakage from immobilized hepatocytes in agarose threads (mean _+S.E., n = 6) before, during and after 1.0 M ethanol perifusion.
The first use of isolated liver ceils for perifusion experiments was described by Van der Meer and Tager [24]. A number of reports on various methods of cell immobilization for perifusion studies using either animal or plant cells for N M R spectroscopy as well as other types of metabolic studies have been published [8-14,25,26]. One method of perifusing isolated human cancer cells for 31p_NMR spectroscopy consists of us-
112 ing gel threads containing a basement membrane material [15]. In the present investigation, a method developed by Foxall et al. [10] has been employed for hepatocyte immobilization in agarosc threads which allows for cell perifusion up to and beyond six hours. This hepatocyte system or bioreactor is readily perifusable under a variety of conditions and is shown herein to be useful as a means of assessing the hepatotoxicity of a wide variety of putative hepatotoxic agents such as AA, ethanol, and AP. As reported by Day ct al. [15], although in vivo studies are probably the most important valuable and interpretable studies, they arc often either difficult or unethical to perform. As a result, a reductionist approach using various cellular systems that simplify by excluding uncontrollable factors present in in vitro situations has been used for assessing drug toxicity and metabolic pathways in vitro. The optimal use of N M R spectroscopy in biology is one in which an investigator is able to correlate spectra obtained in vivo with those obtained in vitro from intact functioning cells in a controlled environment. Within the time frame of the present experiments, the hepatocytes in the perifusion system utilized remained viable as assessed by trypan blue exclusion and LD loss. Moreover, the value for oxygen consumption achieved (21 n m o l / m i n per 106 cells) with this system agrees quite well with reported estimates for oxygen consumption found in the literature for the whole liver which range from 2 - 3 p, m o l / g per min (i.e., approx, equivalent to 20-30 n m o l / m i n per 106 cells), a value achieved in these experiments [27]. Though it is difficult to precisely estimate the number of hepatocytes in a perfused whole liver from which N M R spectra are obtained in a given experiment, following the administration of a fructose load the increase in the PME peak area ratio due to the formation of F-1-P is roughly identical to that obtained when 5 ' 10 s cells are perifused having been immobilized in gel threads. The time-course of the two types of experiment differ, however, because of differences in the diffusion rate of fructose in the agarose-gel matrix to the liver cells and that achieved with whole liver. Nonetheless, the response of the liver cells to a fructose load as seen by 3~P-NMR spectroscopy in both systems is a useful way of assessing liver cell function [28]. The A T P content of isolated perifused hepatocytes was 45 n m o l / 1 0 ~ cells a value which is in good agreement with the value of 3.2-3.8 / x m o l / g or = 48 nmol/106 cells for a freezeclamped whole rat liver [29]. The perifusion of isolated hepatocytes herein reported was similar to what has been applied to other cell types [10,11,25,30,31]. The maintenance of a constant level of /3-ATP during control experiments demonstrates the integrity of this method. Ethanol, AP and the highly hepatotoxic agent, AA, were chosen to test the applicability of the perifusion bioreactor to
investigate the potential in vitro toxicology ul drugs m real time using ~tP_NMR spectroscopx. In lhe experiments performed, no attempt was made to determine the mechanisms for the observed toxicity of the model compounds being studied as each is already reasonably well studied [16-20]. Rather, the agents were chosen for study because they produced a range of hepatotoxicity from minimal to severe, as AP < ethanol < AA. The concentration dependency of the /?/-ATP peak reduction for each of the three model hepatotoxins studied demonstrates the relative hepatotoxicity nf each. The severe toxicity of AA observed with ~ P-NM R can be quantitated by its effect on oxygen consumption and enzyme leakage from cells during the perifusion period. Moreover, it could be compared with studies examining the effect upon liver cell suspensions of exposure to AA at the same concentrations. In the latter situation, a nearly complete loss of LD occurred in 30 min of exposure to AA. Perifusion of hepatocyte threads with AA at the same concentration showed a similar effect only after 80-90 rain with a subsequent progressive increase in the LD leakage and a reduction in the oxygen consumption of the perifused cells with time. In the ethanol experiments, only studies in which the ethanol concentration was considerably higher than those normally used in experiments with isolated liver perfusion or in cells in culture ( > 1.0 M) showed any effect on the system [1,32]. The requirement for a high ethanol concentration at the inflow site of the agarose threads in order to produce cell injury could be the result of any of the following factors: (i) Immobilized cells have greater stability than do non-immobilized cells [9]. This possibility is suggested by the observed relative resistance of perifused cells compared to cell suspensions to the toxic effects of AA, but this could be a concentration effect as well. (ii) The actual concentration of ethanol reaching the cells may be significantly lower than that present in the perifusate. The efficiency of ethanol diffusion into the gel matrix was not estimated in the present experiments. The concentration in the agarose gel at equilibrium and actually reaching the liver cells can be estimated, however, using the diffusion equation for a long cylinder as has been reported by Foxall et al. [10]. Accordingly, the actual concentration in the liver cell would have been expected to be less than that of the perifusate solution per se. Nonetheless, it should be noted that the ethanol concentration of the perifusate was monitored continuously and, at a steady state, it was found that in-flowing and out-flowing concentrations remained constant at 1.0_+ 0.02 M and (I.85 +_0.02 M, respectively. This means that 0.15 M ethanol was extracted by the hepatocyte threads in a single pass. Thus, it appears that the ethanol elimination by the immobilized hepatocytes contained in agarose threads follows a constant rate or
113 zero-order kinetics. Being diffusion limited, the concentration of ethanol actually reaching the cells by necessity must have been less than at the inflow site (1.0 M). Nonetheless, the concentration achieved around the cells would be expected to be considerably greater than the K m of alcohol dehydrogenase present in the rat hepatocytes [33]. Despite a 20% reduction in the /3-ATP signal following ethanol perifusion, the degree of hepatocyte injury judged by the LD leakage was only mild. In addition, although hepatocyte O 2 consumption was reduced by 20% with ethanol perifusion, it increased again after discontinuing the ethanol. The literature on hepatocyte energy metabolism and oxygen consumption under the influence of ethanol is controversial. While some studies have indicated either a constant or reduced amount of A T P in the liver of rats given ethanol acutely [31,34,35], others have suggested that an increased production of A T P following acute treatment of hepatocytes with ethanol occurs [36,37]. Oxygen consumption has been found to be variable following acute ethanol treatment and either increases or decreases depending upon the specific technique or physiological conditions being used [3841]. The results obtained in the present study suggest that a reduction occurs in the total phosphate pool and in the oxygen consumption in response to the toxic effect of ethanol. The observed reduction in the total phosphate pool by ethanol is consistent with results obtained by other authors using whole liver perfusion either with acute ethanol exposure or after chronic ethanol administration [1,2,42]. The Pi peak in the 3~p-NMR spectrum consists of both intracellular and extracellular Pi. Both fructose phosphorylation and ethanol toxicity produce an immediate reduction in the Pi peak suggesting that a significant contribution to the P~ peak is from intracellular Pi" The reduction of Pi with ethanol has been reported previously [1]. It appears that energy status and oxygen consumption of liver cells are reduced but not halted by ethanol as it is reversed by stopping the ethanol perifusion. This is further evidenced by the rather minor increase in LD leakage from cells following ethanol exposure. In cases of severe toxic injury as occurs with AA, the LD levels rise steadily. Finally, the demonstration that the oxygen consumption of the hepatocytes is restored following cessation of the ethanol perifusion confirms the reversibility of the ethanol induced injury. In conclusion, a hepatocyte bioreactor in which cells are immobilized in agarose threads is described. This system allows for liver cell perifusion and viability studies to be performed non-invasively and in real time. Importantly, it provides data that are comparable to those obtained with a perfused isolated liver. Using this model, it has been shown that AP and A A pro-
duced mild and severe hepatotoxicity, respectively. Ethanol perifusion of this system produces a moderate cell injury characterized by an early reduction in the total phosphate pool accompanied by a reduction in oxygen consumption and a slight increase in LD leakage. Each of these is reversed with cessation of the ethanol perifusion. The only residual evidence of ethanol-associated toxicity following cessation of the ethanol perifusion is a reduced A T P level. Based upon these studies, it appears that immobilized hepatocytes are an in vitro system worthy of further evaluation which may prove to be useful in the fields of liver cell metabolism and the response of the liver to putative cytotoxins.
Acknowledgements We wish to thank Ms. Maryann Butowicz and Mr. Andrew D. Laman for their expert technical assistance. We are grateful to the Richard King Mellon Foundation, the Lucille P. Markey Charitable Trust, the Ben Franklin Partnership Program of the Commonwealth of Pennsylvania and the Ralph M. Parsons Foundation for providing financial support for the establishment of the Pittsburgh N M R Center for Biomedical Research. The N M R Center is supported by a grant from the National Institutes of Health (RR-03631). The research described in this p a p e r is supported in part by research grants from the National Institutes of Health (HL-24525 to CH and AA-04425 to DHVT).
References 1 Desmoulin, F., Canioni, P., Crotte, C., Gerolami, A. and Cozzone, P.J. (1987) Hepatology 7, 315-323. 2 Helzberg, J.H., Brown, M.S., Smith, D.J. and Gordon, E.R. (1987) Hepatology 7, 83-88. 3 Cohen, S.M. (1988) in The Liver Biologyand Pathobiology(Arias, M.I., Jakohy, W.B., Popper, H., Schacter, D. and Shafritz, D.A., eds.), pp. 1287-1302, Raven, New York. 4 Iles, R.A., Griffiths, J.R., Stevens, A.N., Gadian, D.G. and Porteous, R. (1980) Biochem. J. 129, 191-202. 5 McLaughlin, A.C., Takeda, H. and Chance, B. (1979) Proc. Natl. Acad. Sci. USA 76, 5445-5449. 6 Murphy, E., Gabel, S.A., Funk, A. and London, R.E. (1988) Biochemistry 27, 526-528. 7 Cohen, S.M., Ogawa, S., Rottenberg, H., Glynn, P., Yamane, T., Brown, T.R. and Shulman, R.G. (1978) Nature 273, 554-556. 8 Gillies, R.J., Chresand, T.J., Drury, P.D. and Dale, B.E. (1986) Rev. NMR Med. 1, 155-179. 9 Vogel, H.J., Brodelins, P., Lilja, H. and Lohmeier-Vogel, E.M. (1987) Methods Enzymol. 135, 512-528. 10 Foxall, D.L., Cohen, J.S. and Mitchell, J.B. (1984) Exp. Cell Res. 154, 521-529. 11 Knop, R.H., Chen, C.W., Mitchell, J.B., Russo, A., McPherson, S. and Cohen, J.S. (1984) Biochim. Biophys. Acta 804, 275-284. 12 Lyon, R.C., Faustino, P.J. and Cohen, J.S. (1986) Magn. Reson. Med. 3, 663-672. 13 Kaplan, O., Aebersold, P. and Cohen, J.S. (1989) FEBS Lett. 258, 55-58.
ll4 14 Van Zijl, P.C.M., Moonen. C.T.W., Faustino, P., Pekar. J., Ka plan, O. and Cohen, J.S. (1991) Proc. Natl. Acad. Sci. USA 88, 3228 3232. 15 Day, P.F., Lyon, R.C., Straka, E.J. and Cohen, J.S. (1988) FASEB J. 2, 2596-2604. 16 Martin, B. (1980) Gastroenterology 78, 382-392. 17 Kitamura, Y., Kamisaki, Y. and Itoh, T. (1989) J. Pharmacol. Exper. Therap. 250, 667-671. 18 Birge, R.B., Bartolone, J.B., Emeigh-Hart, S.G., Nashanian, E.V., Tyson, C.A., Khairallah, E.A. and Cohen, S.D. (1990) Toxicol. Appl. Pharmacol. 105, 472-482. 19 Kyle, M.E., Sakaida, I., Serroni, A. and Farber, J.L. (1990) Biochemical Pharmacol. 40, 1211-1218. 20 Silva J.M. and O'Brien, P.J. (1989) Arch. Biochem. Biophys. 275. 551-558. 21 Moldeus, P., H6gberg, J. and Orrenius, S. (1978) Methods Enzytool. 52, 60-71. 22 Farghali, H., Machkova, Z., Kamenikova, L., Janku, I. and Masek, K. (1984) Meth. Find. Exp. Clin. Pharmacol. 6, 449-454. 23 Campra, J.L. and Reynolds, T.B. (1988) in The Liver Biology and Pathobiology (Arias, M.I., Jakohy, W.B., Popper, H., Schacter, D. and Shafritz, D.A., eds.), pp. 911-930, Raven, New York. 24 Van der Meer, R. and Tager, J.M. (1976) FEBS Lett. 67, 36-40. 25 Foxall, D.H. and Cohen, J.S. (1983) J. Magn. Res. 52, 346-349. 26 Hulst, A.C., Tramper, J., Brodelius, P., Eijkenboom, L.J.C. and Layben, K.Ch.A.M. (1985) J. Chem. Tech. Biotechnol. 35B, 198204. 27 Younes, M., Wagner, H. and Strubelt, O. (1989) Biochem. Pharmacol. 38, 3573-3581.
28 Terrier, F., Vock, P., ('otting, J., Ladebeck, R., Rcichcn. 1 and HentscheI, D.(1989) Radiology 171, 557 5(~3. 29 Sakr, M.F.. Zelti. G.M., Hassnein, T.I., Farghali, tt., Nalcsnik, M.A., Gavaler, J.S., Starzl. T.E. and Van Thiel. I).11. (19911 Hepatology 13, 947 951. 30 Brindle, K. and Krikler, S. (1985) Biochim. Biophys. Acla 847, 285-292. 31 Santos, H. and Turner, D.L (1986)J. Magn. Reson. (3S, 345-349. 32 Voci, A., Gallo, G., Balestero, F. and Fugassa, F]. (19881 ('ell Biol. Int. Rpts. 12. 647-660. 33 Goldstein, D.B. (1983) Pharmacology of Alcohol, Oxford University Press, New York. 34 Hernandez-Munoz, R., Santamaria, A.. Garcia-Sainz. J.A., Pina, E. and De Sanchez, C. (1978) Arch. Biochem. Biophys. 190, 155-162. 35 Gillam, E. and Ward, L.C. (1986) Int. J. Biochem. 18, 1031-1038. 36 French, S.W. (1966) Proc. Soc. Exp. Biol. Med. 121,681-689. 37 Baranyai, J.M. and Blum, J.J. (1989) Biochem. J. 258, 121-140. 38 Thurman, R.G. and Scholz, R. (1977) Eur. J. Biochem. 75, 13-21. 39 Yuki, T., Thurman, R.G., Schwabe. U. and Scholz. R. (1980) Biochem. J. 186, 997 1000. 40 Yuki, T., Israel, Y. and Thurman, R.G. (1982) Biochem. Pharmacol. 31, 2403-24(17. 41 Stowell, K.M. and Crow, K.E. (1985) Biochem. J. 262, 595-6(12. 42 Takahashi, H., Geoffrion, Y., Butler, K.W. and French, S.W. (1990) Hepatotogy I 1, 65 73.
105
BBADIS 61158
31p_NMR spectroscopy of perifused rat hepatocytes immobilized in agarose threads: application to chemical-induced hepatotoxicity Hassan Farghali a, Lorenzo Rossaro a, Judith S. Gava]er b, David H. Van Thiel ~', Susan R. Dowd c, D o n a l d S. Williams d and Chien Ho c,d " Departments of Surgery, Unicersity of Pittsburgh School of Medicine, Pittsburgh, PA (USA), t, Department of Medicine, Unirersity of Pittsburgh School of Medicine, Pittsburgh, PA (USA), ~'Department of Biological Sciences, Carnegie Mellon Unicersity, Pittsburgh, PA (USA), and ,I Pittsburgh NMR Center for Biomedical Research, Carnegie Mellon Unirersity, Pittsburgh, PA (USA) (Received 12 December 1991)
Key words: NMR, 31p_; Hepatocyte; Hepatotoxicity; Ethanol; Acetaminophen; Allyl alcohol; Fructose; Agarose thread
A system consisting of isolated rat hepatocytes immobilized in agarose threads continuously perifused with oxygenated Krebs-Henseleit (KH) solution has been found to maintain cell viability with excellent metabolic activity for more than 6 h. The hepatocytes were monitored by phosphorus-31 nuclear magnetic resonance (3~p_NMR) spectroscopy at 4.7 Tesla, by measurement of oxygen consumption and by the leakage of lactate dehydrogenase (LD) and alanine aminotransferase (ALT). The data obtained were comparable to those found for an isolated perfused whole liver in vitro. The effects of allyl alcohol (AA), ethanol, and 4-acetaminophenol lAP) were examined. A solution of 225 IzM AA perifused for 90 min caused the disappearance of the /3-phosphate resonance of adenosine triphosphate (ATP) in the 3]P_NMR spectra, a 7-fold increase in LD leakage and a 70% reduction in oxygen consumption. Ethanol (1.0 M) perifused for 90 min reduced the /3-ATP signal intensity ratio by 20%, the phosphomonoester (PME) signal by 50% and inorganic phosphate (Pi) by 33% (P < 0.05). AP (10 mM) caused only mild liver-cell damage. The results demonstrate that perifused immobilized hepatocytes can be used as a liver model to assess the effects of a wide range of chemicals and other xenobiotics by NMR spectroscopy.
Introduction The application of nuclear magnetic resonance (NMR) spectroscopy to investigate metabolic parameters affecting hepatic function under different pathophysiological conditions has been documented during the past 11 years [1-6]. Few N M R spectroscopic studies, however, have been carried out on isolated liver cells [7]. N M R spectroscopic studies of isolated liver cells offer the advantage of precise control over the sample and the accuracy of quantitative measurements which can be carried out on a known type and number of ceils [8]. This is particularly important when studying
Abbreviations: KH, Krebs-Henseleit; 31p-NMR, phosphorus-31 nuclear magnetic resonance; LD, lactate dehydrogenase; ALT, alanine aminotransferase; AP, 4-acetaminophenol; ATP, adenosine triphosphate; AA, allyl alcohol; PME, phosphomonoester; Pi, inorganic phosphate. Correspondence: C. Ho, Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA, 15213-2683, USA.
the physiology and toxicology of cells non-invasively. To acquire meaningful information, an efficient perifusion bioreactor model needs to be used which allows for the provision of nutrients to the cells with and without drugs, the removal of cellular waste products, adequate oxygenation and the prevention of cell injury. In addition, a critical requirement for N M R studies is a relatively densely-packed cell suspension to obtain a spectrum with a reasonable signal-to-noise ratio and spectral resolution. Gillies et al. [8] have described various methods of cell immobilization and their application to cell biology including studies utilizing NMR. It is well recognized that immobilized cells have a greater stability than do non-immobilized cells [9]. The metabolite patterns of immobilized Chinese hamster lung fibroblasts have been monitored over many hours using 3~p-NMR [10,11] as well as pooled human lymphocytes [12] using N M R and 13C-labeled metabolites. 3~p-NMR studies have been applied to perifused interleukin-2-activated lymphocytes and tumor-infiltrating lymphocytes [13]. In these studies, it has been possible to separate intra-
106
cellular and extracellular information from the NMR spectra obtained for the perifused cells [14]. The method herein reported involves the application of real-time 3~p-NMR spectroscopy to the study of isolated hepatocytes immobilized in low-gelling agarose threads [10,15]. The metabolic behavior of isolated hepatocytes maintained in agarose gel was assessed under basal conditions and was compared to that obtained using a perfused whole liver. The response to allyl alcohol (AA), ethanol and 4-acetaminophenol (AP) perifusion at various concentrations and durations of exposure was also investigated. The compounds were chosen for their known ability to produce hepatic injuries which range from mild to severe [16-20]. Due to the medical and socioeconomic importance of alcohol, ethanol was chosen to be studied in the greatest detail. Additional conventional biochemical procedures were performed whenever possible. Materials and Methods
Sea plaque agarose (low-gelling t e m p e r a t u r e agarose) was obtained from FMC (Rockland, ME). Ethylene glycol-bis (/3-aminoethylether) N,N,N',N'-tetraacetic acid (EGTA), 4-acetaminophenol (AP) (acetaminophen), trypan blue, bovine serum albumin (BSA), methylenediphosphonic acid (MDPA) and diagnostic kits for the measurement of lactate dehydrogenase (LD) and adenosine-5'-triphosphate (ATP) were obtained from Sigma (St. Louis, MO). Collagenase was obtained from SEVAC (Institute Sera and Vaccines Prague). Allyl alcohol (AA) was obtained from Aldrich Chemical Company (Milwaukee, WI). Ethyl alcohol (ethanol), fructose and all other chemicals were reagent grade and were obtained from standard sources. Thin-
OUTSIDE THE MAGNET
Reservoir
wall Chem fluor T F E tubing (I.D. ().(12(~ in) was o b tained from B e r g h o f / A m e r i c a (Concord, (?A),
Preparation of the isolated hepatocytes Isolated hepatocytes were prepared using a twophase perfusion technique with the collagenase (0.67 m g / m l ) being added in the second phase as reported earlier [21,22]. After isolating and counting the recovered liver cells, viability was assessed both biochemically by LD leakage and histologically by trypan blue exclusion. Only cell preparations that excluded trypan blue to the extent of more than 95% were used. Thc cell density in all experiments was 4-5 - 1(17 cells/ml.
Immobilization of hepatocytes in agarose threads The 1.8% low-gelling agarose solution was prepared in warm Krebs-Henseleit (KH) solution (NaC1 6.99 g, KC1 0.36 g, K H 2 P O 4 0.13 g, MgSO 4- 7 H~O 0.295 g, C a C 1 2 . H 2 0 0.37, NaHCO 3 2 g per liter, pH 7.4) at 70°C. The agarose solution was brought to 37°C. The thermostated agarose solution was mixed 1:1 with hepatocytes which were isolated from fed SpragueDawley male rats, at a density of 4 - 5 . 107 cells/ml. The cells were immobilized in agarose threads by extruding the agarose-cell mixture through cooled Chem fluor T F E tubing into a 3-cm outer diameter NMR tube containing KH solution under carbogen (95% 0 2 and 5% CO:). The specific details of this technique have been described previously by Foxall et al. [10]. The threads were compressed gently to a final vol. of 20 ml which was entirely covered by the NMR coil. A solution of MDPA (100 retool, pH 9.5) sealed in a spherical glass bulb and positioned within the threads served as an external reference for both ~Lp chemical shifts and relative concentration determinations.
INSIDE THE MAGNET HeQt ExchQnger
Pump2~
NMR Probe
Filter
Thermostat i
\
/
t
GOS Mixture
,,
RF Coil
Outflow
i
Fig. 1. A schematic representation of the perifusion system of the hepatocytes immobilized in agarose threads. The left side represents a thermoregulated and oxygenated perifusion fluid flowing at 10 m l / m i n by p u m p 1. Pump 2 is connected to the perifusion system by a three-way stopcock to deliver the alcohol. The right side illustrates the interior of the magnet bore where a heat exchanger enables the inflow perifusate to be heated prior to flowing into the N M R tube.
107
Perifusion of the threads: 31p-NMR spectroscopy, viability testing and biochemical analyses A schematic representation of the perifusion system for the hepatocytes immobilized in the agarose threads is shown in Fig. 1. The threads were perifused with a thermoregulated and well-oxygenated (95% 0 2 and 5% CO 2) K H medium at a rate of 10 m l / m i n in a non-recirculating system using a roller p u m p (pump 1). This perifusion rate was selected on the basis of its ability to maintain a high A T P level within the hepatocytes. In addition, it is known that under physiologic conditions, hepatic blood flow ranges from 100-130 m l / m i n per 100 g [23]. A second p u m p (pump 2) was used to deliver the compound of interest to the perifusate at a predetermined concentration using a threeway stopcock without affecting the final flow rate. Before initiating perifusion with the drug, the threads were equilibrated until a constant 3~P-NMR baseline spectrum and oxygen consumption rate were observed (about 60 min). These measurements were continued for a total of 6 h in the initial experiments to demonstrate that the hepatocytes immobilized in agarose threads were viable and stable for periods equal to those to be used in all subsequent experiments. Immobilized liver cell viability and function were assessed periodically by: (i) measuring 0 2 consumption calculated from rttPo2 , inflow outflow', blank -Po2 )-Po2 ]- flow rate; (ii) monitoring the percent of total LD leakage from cells at regular intervals for more than 6 h compared to the maximum lysate LD activity; (iii) measuring the fructose phosphorylation achieved after a fructose bolus; and (iv) measuring cytologically, in a separate experiment, the trypan blue (0.5%) exclusion after 6 h of perfusion. The A A used was diluted with K H solution to achieve a final concentration in the inflow perifusate of 100, 225 or 500/.tM. The AP was dissolved in ethanol and then diluted with K H solution to produce a final concentration of 2.5, 5 or 10 mM. In the AP experiments, the maximum ethanol concentration used to solubilize the AP in K H perifusate was 165 raM. Previous experiments have shown that this concentration of ethanol does not affect either the 31p-NMR spectra or the biochemical p a r a m e t e r s of the hepatocytes (results not shown). Ethanol was diluted with K H solution to provide a final concentration of 0.50, 0.85, 1.0 or 1.2 M in the perifusate. The actual concentrations obtained at initial exposure to the cells at the inflow and the outflow sites were determined at regular intervals as described in the Results section. Preliminary experiments showed that perifusion of an ethanol solution for 90 min followed by a wash-out with K H solution for 27 min gave highly reproducible results. The functional integrity of the cells was determined by measuring the oxygen consumption, LD and A L T leakage, as well as the fructose phosphorylation
response. The fructose studies consisted of perifusing the ceils with fructose in K H solution at a rate of 1.5 m m o l / m l per min delivered as a 1-min bolus. The area of the fructose-l-phosphate (F-l-P) signal in the 31p_ N M R spectrum was calculated for both control and ethanol perifused cells. In most cases, samples were obtained every 20 min from the outflow perifusate for various biochemical and oxygen consumption analyses, both during and after specific compound perifusion. Oxygen consumption was determined using an ABL2 acid-base laboratory instrument (Radiometer, Copenhagen). The results were calculated as n m o l / m i n per 106 hepatocytes. All other biochemical parameters such as LD leakage, /3-ATP, phosphomonoester (PME) and inorganic phosphate (Pi), were normalized to values achieved with 5 • 10 ~ cells. 3~p-NMR spectra were obtained at 81 MHz with a 3-cm solenoid coil at 37°C. Spectra were continuously recorded in blocks of 256 scans accumulated at a repetition rate of 2 s using a Bruker Biospec 1I spectrometer operating at 4.7 Tesla and equipped with a 40-cm bore-sized, horizontal superconducting solenoid. The areas under the peaks were determined using the integration routine available with the Bruker software. All signal areas reported are relative to the 31P-NMR reference compound, MDPA.
Functionality comparison with isolated perfused liver The abdomen of heparinized rats (500 U / r a t ) was entered via a mid[ine laparotomy and the infra-renal aorta was cannulated using a 20 G catheter. The liver was perfused in situ with 30 ml of K H solution after incising the inferior vena cava above and below the liver and clamping the aorta proximal to the celiac axis. The portal vein was cannulated using a second 20-G catheter whereby the liver was continuously perfused via the portal vein and the aorta with KH solution while the liver was being excised. The time from aortic clamping and initiation of the hepatic perifusion was less than 10 min in all cases. Perfusion of the liver was accomplished as described for thread perifusion. A bulb containing an external M D P A reference was included in the N M R sample tube.
Enzymatic evaluation and ATP in hepatocyte threads and whole liver Hepatocyte threads were prepared, perifused with K H solution for 1 h as described and then freeze clamped in liquid nitrogen. A portion of the frozen threads or about 1 g of livers obtained from rats under ether anaesthesia was ground in liquid nitrogen and 4% ( w / v ) pre-cooled perchloric acid (1:5, w / v ) and homogenized. The resultant homogenized samples were centrifuged at 4°C at 3000 rpm for 10 min. The supernatants were brought to p H 7 using 6 M K2CO 3 and
108 energy status for up to 6 h is shown in Fig. 2. In contrast, h e p a t o c y t c s isolated without t h r e a d i n g and p e r i f u s i o n s h o w e d no 3~P-NMR signals (results not shown). T h e viability o f p e r i f u s e d cells for p e r i o d s tip
4 A
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Fig. 2. ~IP-NMR spectra of immobilized perifused rat hepatocytes over a 6-h period at 37°C. I and It are spectra obtained after perifusion of 0 and 6 h, respectively. Peak assignments: (1) 13-phosphate of ATP; (2) oPphosphate of ATP; (3) y-phosphate of ATP; (4) inorganic phosphate (Pi); and (5) phosphomonoesters (PME). r e s p u n at 4°C at 3000 r p m a n d k e p t at - 7 0 ° C until assayed s p e c t r o p h o t o m e t r i c a l l y for t h e i r A T P c o n t e n t .
i
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10
Allyl alcohol induced hepatotoxicity in non-perifused isolated hepatocytes O n l y A A was t e s t e d using n o n - p e r i f u s e d i s o l a t e d h e p a t o c y t e s b e c a u s e it has t h e g r e a t e s t h e p a t o t o x i c i t y . H e p a t o c y t e s w e r e i n c u b a t e d for 20 rain at a c o n c e n t r a tion of 2 • 106 c e l l s / m i in a plastic t u b e at 37°C in K H solution at p H 7.4 u n d e r an a t m o s p h e r e of 95% O 2 a n d 5 % COz b e f o r e the a d d i t i o n o f A A at a c o n c e n t r a tion of 225 tzM. E x p e r i m e n t s using c o n t r o l u n t r e a t e d h e p a t o c y t e s w e r e run in parallel. Five r e p l i c a t e experim e n t s using d i f f e r e n t rats w e r e u s e d for e a c h t r e a t ment. A l i q u o t s w e r e o b t a i n e d at 0, 30, 60 a n d 120 rain after the A A a d d i t i o n for L D l e a k a g e m e a s u r e m e n t . E n z y m e l e a k a g e was m o n i t o r e d b o t h in an aliquot of cell-free m e d i u m a n d a f t e r lysis o f the cells with l % T r i t o n X-100. T h e l e a k a g e of e n z y m e u n d e r experim e n t a l c o n d i t i o n s was e x p r e s s e d as a p e r c e n t of the total lysate activity.
Statistical analysis A n a l y s i s of v a r i a n c e s with t h e T u k e y m u l t i p l e comp a r i s o n p r o c e d u r e was u s e d to e v a l u a t e c h a n g e s in r e c o r d e d v a r i a b l e s over time.
Results T h e ability o f h e p a t o c y t e s within the g e l - t h r e a d p e r ifusion system to r e a c h a n d m a i n t a i n a stable c e l l u l a r
i
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Fig. 3. (A) 31P-NMR spectra of immobilized rat hepatocytes at 37°C. After hepatocyte stabilization, fructose (F) was perifused at a rate of 1.5 mmol/ml/min in KH solution for 1 min. Spectra I - I l l were obtained I~efore, 14 and 32 min, respectively, after fructose perifusion. (B) 3~p-NMR spectra of perfused isolated rat liver at 37°C before (I) and 8 min after (ll) a fructose challenge. The immediate appearance of fructose-l-phosphate (peak 5) and its gradual disappearance over time is evident. The peak assignments are the same as in Fig. 2 (peak 6 is MDPA, the external reference).
109
to 6 h as assessed by trypan blue exclusion was 95%. The large Pi signal recorded in the 31P-NMR spectrum is due to the phosphate buffer as both the intra- and extracellular Pi contribute to the recorded Pi signal. The administration of a fructose load resulted in a sharp reduction in the Pi signal, suggesting that the P~ peak is dominated by intracellular Pi, a concomitant reduction in the ATP signal and a significant increase in the PME signal due to the accumulation of F-1-P as shown in Fig. 3A. Following the administration of a fructose challenge to an isolated perfused liver (Fig. 3B), a similar increase in the PME area was observed immediately with an associated reduction in the Pi and
4000" A 300O" E :D
2000"
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Fig. 5. Time-course of 225 /xM A A on ( A ) L D leakage and (B) the oxygen consumption of perifused immobilized hepatocytes in agarose threads. Time 0 represents the start of A A perifusion. (mean + S.E., I
II
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Fig. 4. 31p-NMR spectra of immobilized rat hepatocytes perifused with 225 g M A A at different time intervals at 37°C. Spectra I - V I are control, and 18, 50, 68, 80 a n d 90 min after A A perifusion, respectively.
n = 3).
ATP content of the liver. In several experiments with either KH-perifused cell gel threads or perfused whole liver, a 2-fold increase in PME due to F-1-P was seen. In both cases, 0 2 consumption and LD release were similar for several hours of perifusion. Hepatocytes in gel threads perifused with KH up to 6 h exhibited continued trypan blue exclusion (90%). The ATP content measured by enzymatic methods for perifused hepatocyte threads and whole liver was 45 nmol/106 cells and 3.8 ixmol/g, respectively, on average. Perifusion with A A produced measurable reductions in the phosphate pool at concentrations of 225 txM and above (results not shown). Fig. 4 shows the time-course for the 3~P-NMR spectra of the hepatocyte threads perifused with AA. A reduction in the ATP peaks was evident after 80 rain. A significant increase in the LD leakage and an induced reduction in 0 2 consumption were also seen (Fig. 5A and B). When isolated hepatocytes were incubated with 225 /xM AA, essentially
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Fig. 6. The time-course of 225 ~xM AA on the percent leakage of LD from isolated liver cell incubations. Time 0 represents the start of AA perifusion (mean + S . E . , n = 3 ) . * indicates significantly different (P < 0.05).
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100% LD leakage was obtained after 30 min of expos u r e (Fig. 6). A s e x p e c t e d , o n l y a v e r y f e w r e s i d u a l c e l l s w e r e a b l e t o e x c l u d e t r y p a n b l u e in t h e s e s t u d i e s . ethanol
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Fig. 7 3~p_NMR spectra of immobilized rat bepatocytes perifused at different ethanol concentrations at 37°C. I (control), II, II1 and IV are after perifusion with 0.85, 1.0 and 1.2 M ethanol, respectively. The peak assignments are the same as Fig. 2 (Peak 6 is the signal from the external standard MDPA).
F 200
(min)
Fig. 8. The time-course of signal intensity ratios (mean _+S.E., n = 6): A, /3-ATP/MDPA; B, P M E / M D P A ; and C, P i / M D P A under the influence of 1.0 M ethanol perifusion for 90 rain. After ethanol perifusion and a washout, fructose (F) was perifused as a 1,5 mmol/ml per min bolus for 1 rain. The effect of ethanol at various concentrations on the 3Ip-NMR spectra obtained from the immobilized h e p a t o c y t e s is s h o w n in Fig. 7. A f t e r o b t a i n i n g s t a b l e
111 baseline spectra, the threads were perifused with 0.85, 1.0 or 1.2 M ethanol for 30 min followed by a 30-min washout period. These higher ethanol concentrations were selected because cells exposed to 0.50 M ethanol for 4 h did not show any change in their 31p-NMR (results not shown). From Fig. 7, a concentration-dependent decline in ATP and Pi with ethanol is evident• In order to study the detailed time-course of the effect of ethanol on cellular energy parameters, perifusion with 1.0 M ethanol was maintained for 90 min and the time-course of the /3-ATP, PME and Pi signal intensities were followed (Fig. 8A, B and C, respectively). Perifusion at this ethanol concentration resulted in a decline of about 20% in the /3-ATP level which plateaued and was statistically lower than the level during the subsequent perifusion period ( P < 0.05). After a 30-min washout with KH solution followed by a bolus of fructose, a further decline in t h e / 3 - A T P ratio occurred, followed by a slow recovery of the ATP signal to the pre-fructose level, but not to the preethanol basal level. The PME signals over these same six experiments showed a gradual decline with a minimum level achieved only at the end of the ethanolperifusion period. The administration of the fructose bolus was associated with a significant, sharp rise in the PME signal followed by a slower decrease to the prefructose level. The P~ signal followed the same pattern as the /3-ATP signal. Analyses of the perifusate for the cytosolic enzyme, LD, gave the results illustrated in Fig. 9. Ethanol perifusion produced a gradual but transient increase in
500"
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~6o Time
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Fig. 10. Time-course of oxygen consumption by immobilized hepatocytes in agarose threads (mean _+S.E., n = 6) before, during and after 1.0 M ethanol perifusion.
the level of enzyme in the perifusate for about 60 min, at which time the levels were significantly greater than those of the controls ( P < 0.05). Oxygen consumption was measured before, during, and after ethanol perifusion. The results are shown in Fig. 10. Under basal conditions, the immobilized cells consumed a mean of 21 nmol O 2 / m i n per 106 cells. Ethanol perifusion for 60 min reduced the 0 2 consumption significantly ( P < 0.05). After discontinuing the ethanol perifusion, O 2 consumption levels recovered slightly. The effect of different concentrations of AP on the 31p-NMR spectra obtained for the immobilized hepatocytes has been examined (results not shown). Only after 70 min of perifusion with 10 mM AP did a slight reduction occur in the /3-ATP peak resonance. This was accompanied by a slight increase in the LD leak and a slight decrease in the oxygen consumption at the highest concentration and duration of exposure.
.J
Discussion 100
0
0
160 Time
280
360
(min)
Fig. 9. Time-course of LD leakage from immobilized hepatocytes in agarose threads (mean _+S.E., n = 6) before, during and after 1.0 M ethanol perifusion.
The first use of isolated liver ceils for perifusion experiments was described by Van der Meer and Tager [24]. A number of reports on various methods of cell immobilization for perifusion studies using either animal or plant cells for N M R spectroscopy as well as other types of metabolic studies have been published [8-14,25,26]. One method of perifusing isolated human cancer cells for 31p_NMR spectroscopy consists of us-
112 ing gel threads containing a basement membrane material [15]. In the present investigation, a method developed by Foxall et al. [10] has been employed for hepatocyte immobilization in agarosc threads which allows for cell perifusion up to and beyond six hours. This hepatocyte system or bioreactor is readily perifusable under a variety of conditions and is shown herein to be useful as a means of assessing the hepatotoxicity of a wide variety of putative hepatotoxic agents such as AA, ethanol, and AP. As reported by Day ct al. [15], although in vivo studies are probably the most important valuable and interpretable studies, they arc often either difficult or unethical to perform. As a result, a reductionist approach using various cellular systems that simplify by excluding uncontrollable factors present in in vitro situations has been used for assessing drug toxicity and metabolic pathways in vitro. The optimal use of N M R spectroscopy in biology is one in which an investigator is able to correlate spectra obtained in vivo with those obtained in vitro from intact functioning cells in a controlled environment. Within the time frame of the present experiments, the hepatocytes in the perifusion system utilized remained viable as assessed by trypan blue exclusion and LD loss. Moreover, the value for oxygen consumption achieved (21 n m o l / m i n per 106 cells) with this system agrees quite well with reported estimates for oxygen consumption found in the literature for the whole liver which range from 2 - 3 p, m o l / g per min (i.e., approx, equivalent to 20-30 n m o l / m i n per 106 cells), a value achieved in these experiments [27]. Though it is difficult to precisely estimate the number of hepatocytes in a perfused whole liver from which N M R spectra are obtained in a given experiment, following the administration of a fructose load the increase in the PME peak area ratio due to the formation of F-1-P is roughly identical to that obtained when 5 ' 10 s cells are perifused having been immobilized in gel threads. The time-course of the two types of experiment differ, however, because of differences in the diffusion rate of fructose in the agarose-gel matrix to the liver cells and that achieved with whole liver. Nonetheless, the response of the liver cells to a fructose load as seen by 3~P-NMR spectroscopy in both systems is a useful way of assessing liver cell function [28]. The A T P content of isolated perifused hepatocytes was 45 n m o l / 1 0 ~ cells a value which is in good agreement with the value of 3.2-3.8 / x m o l / g or = 48 nmol/106 cells for a freezeclamped whole rat liver [29]. The perifusion of isolated hepatocytes herein reported was similar to what has been applied to other cell types [10,11,25,30,31]. The maintenance of a constant level of /3-ATP during control experiments demonstrates the integrity of this method. Ethanol, AP and the highly hepatotoxic agent, AA, were chosen to test the applicability of the perifusion bioreactor to
investigate the potential in vitro toxicology ul drugs m real time using ~tP_NMR spectroscopx. In lhe experiments performed, no attempt was made to determine the mechanisms for the observed toxicity of the model compounds being studied as each is already reasonably well studied [16-20]. Rather, the agents were chosen for study because they produced a range of hepatotoxicity from minimal to severe, as AP < ethanol < AA. The concentration dependency of the /?/-ATP peak reduction for each of the three model hepatotoxins studied demonstrates the relative hepatotoxicity nf each. The severe toxicity of AA observed with ~ P-NM R can be quantitated by its effect on oxygen consumption and enzyme leakage from cells during the perifusion period. Moreover, it could be compared with studies examining the effect upon liver cell suspensions of exposure to AA at the same concentrations. In the latter situation, a nearly complete loss of LD occurred in 30 min of exposure to AA. Perifusion of hepatocyte threads with AA at the same concentration showed a similar effect only after 80-90 rain with a subsequent progressive increase in the LD leakage and a reduction in the oxygen consumption of the perifused cells with time. In the ethanol experiments, only studies in which the ethanol concentration was considerably higher than those normally used in experiments with isolated liver perfusion or in cells in culture ( > 1.0 M) showed any effect on the system [1,32]. The requirement for a high ethanol concentration at the inflow site of the agarose threads in order to produce cell injury could be the result of any of the following factors: (i) Immobilized cells have greater stability than do non-immobilized cells [9]. This possibility is suggested by the observed relative resistance of perifused cells compared to cell suspensions to the toxic effects of AA, but this could be a concentration effect as well. (ii) The actual concentration of ethanol reaching the cells may be significantly lower than that present in the perifusate. The efficiency of ethanol diffusion into the gel matrix was not estimated in the present experiments. The concentration in the agarose gel at equilibrium and actually reaching the liver cells can be estimated, however, using the diffusion equation for a long cylinder as has been reported by Foxall et al. [10]. Accordingly, the actual concentration in the liver cell would have been expected to be less than that of the perifusate solution per se. Nonetheless, it should be noted that the ethanol concentration of the perifusate was monitored continuously and, at a steady state, it was found that in-flowing and out-flowing concentrations remained constant at 1.0_+ 0.02 M and (I.85 +_0.02 M, respectively. This means that 0.15 M ethanol was extracted by the hepatocyte threads in a single pass. Thus, it appears that the ethanol elimination by the immobilized hepatocytes contained in agarose threads follows a constant rate or
113 zero-order kinetics. Being diffusion limited, the concentration of ethanol actually reaching the cells by necessity must have been less than at the inflow site (1.0 M). Nonetheless, the concentration achieved around the cells would be expected to be considerably greater than the K m of alcohol dehydrogenase present in the rat hepatocytes [33]. Despite a 20% reduction in the /3-ATP signal following ethanol perifusion, the degree of hepatocyte injury judged by the LD leakage was only mild. In addition, although hepatocyte O 2 consumption was reduced by 20% with ethanol perifusion, it increased again after discontinuing the ethanol. The literature on hepatocyte energy metabolism and oxygen consumption under the influence of ethanol is controversial. While some studies have indicated either a constant or reduced amount of A T P in the liver of rats given ethanol acutely [31,34,35], others have suggested that an increased production of A T P following acute treatment of hepatocytes with ethanol occurs [36,37]. Oxygen consumption has been found to be variable following acute ethanol treatment and either increases or decreases depending upon the specific technique or physiological conditions being used [3841]. The results obtained in the present study suggest that a reduction occurs in the total phosphate pool and in the oxygen consumption in response to the toxic effect of ethanol. The observed reduction in the total phosphate pool by ethanol is consistent with results obtained by other authors using whole liver perfusion either with acute ethanol exposure or after chronic ethanol administration [1,2,42]. The Pi peak in the 3~p-NMR spectrum consists of both intracellular and extracellular Pi. Both fructose phosphorylation and ethanol toxicity produce an immediate reduction in the Pi peak suggesting that a significant contribution to the P~ peak is from intracellular Pi" The reduction of Pi with ethanol has been reported previously [1]. It appears that energy status and oxygen consumption of liver cells are reduced but not halted by ethanol as it is reversed by stopping the ethanol perifusion. This is further evidenced by the rather minor increase in LD leakage from cells following ethanol exposure. In cases of severe toxic injury as occurs with AA, the LD levels rise steadily. Finally, the demonstration that the oxygen consumption of the hepatocytes is restored following cessation of the ethanol perifusion confirms the reversibility of the ethanol induced injury. In conclusion, a hepatocyte bioreactor in which cells are immobilized in agarose threads is described. This system allows for liver cell perifusion and viability studies to be performed non-invasively and in real time. Importantly, it provides data that are comparable to those obtained with a perfused isolated liver. Using this model, it has been shown that AP and A A pro-
duced mild and severe hepatotoxicity, respectively. Ethanol perifusion of this system produces a moderate cell injury characterized by an early reduction in the total phosphate pool accompanied by a reduction in oxygen consumption and a slight increase in LD leakage. Each of these is reversed with cessation of the ethanol perifusion. The only residual evidence of ethanol-associated toxicity following cessation of the ethanol perifusion is a reduced A T P level. Based upon these studies, it appears that immobilized hepatocytes are an in vitro system worthy of further evaluation which may prove to be useful in the fields of liver cell metabolism and the response of the liver to putative cytotoxins.
Acknowledgements We wish to thank Ms. Maryann Butowicz and Mr. Andrew D. Laman for their expert technical assistance. We are grateful to the Richard King Mellon Foundation, the Lucille P. Markey Charitable Trust, the Ben Franklin Partnership Program of the Commonwealth of Pennsylvania and the Ralph M. Parsons Foundation for providing financial support for the establishment of the Pittsburgh N M R Center for Biomedical Research. The N M R Center is supported by a grant from the National Institutes of Health (RR-03631). The research described in this p a p e r is supported in part by research grants from the National Institutes of Health (HL-24525 to CH and AA-04425 to DHVT).
References 1 Desmoulin, F., Canioni, P., Crotte, C., Gerolami, A. and Cozzone, P.J. (1987) Hepatology 7, 315-323. 2 Helzberg, J.H., Brown, M.S., Smith, D.J. and Gordon, E.R. (1987) Hepatology 7, 83-88. 3 Cohen, S.M. (1988) in The Liver Biologyand Pathobiology(Arias, M.I., Jakohy, W.B., Popper, H., Schacter, D. and Shafritz, D.A., eds.), pp. 1287-1302, Raven, New York. 4 Iles, R.A., Griffiths, J.R., Stevens, A.N., Gadian, D.G. and Porteous, R. (1980) Biochem. J. 129, 191-202. 5 McLaughlin, A.C., Takeda, H. and Chance, B. (1979) Proc. Natl. Acad. Sci. USA 76, 5445-5449. 6 Murphy, E., Gabel, S.A., Funk, A. and London, R.E. (1988) Biochemistry 27, 526-528. 7 Cohen, S.M., Ogawa, S., Rottenberg, H., Glynn, P., Yamane, T., Brown, T.R. and Shulman, R.G. (1978) Nature 273, 554-556. 8 Gillies, R.J., Chresand, T.J., Drury, P.D. and Dale, B.E. (1986) Rev. NMR Med. 1, 155-179. 9 Vogel, H.J., Brodelins, P., Lilja, H. and Lohmeier-Vogel, E.M. (1987) Methods Enzymol. 135, 512-528. 10 Foxall, D.L., Cohen, J.S. and Mitchell, J.B. (1984) Exp. Cell Res. 154, 521-529. 11 Knop, R.H., Chen, C.W., Mitchell, J.B., Russo, A., McPherson, S. and Cohen, J.S. (1984) Biochim. Biophys. Acta 804, 275-284. 12 Lyon, R.C., Faustino, P.J. and Cohen, J.S. (1986) Magn. Reson. Med. 3, 663-672. 13 Kaplan, O., Aebersold, P. and Cohen, J.S. (1989) FEBS Lett. 258, 55-58.
ll4 14 Van Zijl, P.C.M., Moonen. C.T.W., Faustino, P., Pekar. J., Ka plan, O. and Cohen, J.S. (1991) Proc. Natl. Acad. Sci. USA 88, 3228 3232. 15 Day, P.F., Lyon, R.C., Straka, E.J. and Cohen, J.S. (1988) FASEB J. 2, 2596-2604. 16 Martin, B. (1980) Gastroenterology 78, 382-392. 17 Kitamura, Y., Kamisaki, Y. and Itoh, T. (1989) J. Pharmacol. Exper. Therap. 250, 667-671. 18 Birge, R.B., Bartolone, J.B., Emeigh-Hart, S.G., Nashanian, E.V., Tyson, C.A., Khairallah, E.A. and Cohen, S.D. (1990) Toxicol. Appl. Pharmacol. 105, 472-482. 19 Kyle, M.E., Sakaida, I., Serroni, A. and Farber, J.L. (1990) Biochemical Pharmacol. 40, 1211-1218. 20 Silva J.M. and O'Brien, P.J. (1989) Arch. Biochem. Biophys. 275. 551-558. 21 Moldeus, P., H6gberg, J. and Orrenius, S. (1978) Methods Enzytool. 52, 60-71. 22 Farghali, H., Machkova, Z., Kamenikova, L., Janku, I. and Masek, K. (1984) Meth. Find. Exp. Clin. Pharmacol. 6, 449-454. 23 Campra, J.L. and Reynolds, T.B. (1988) in The Liver Biology and Pathobiology (Arias, M.I., Jakohy, W.B., Popper, H., Schacter, D. and Shafritz, D.A., eds.), pp. 911-930, Raven, New York. 24 Van der Meer, R. and Tager, J.M. (1976) FEBS Lett. 67, 36-40. 25 Foxall, D.H. and Cohen, J.S. (1983) J. Magn. Res. 52, 346-349. 26 Hulst, A.C., Tramper, J., Brodelius, P., Eijkenboom, L.J.C. and Layben, K.Ch.A.M. (1985) J. Chem. Tech. Biotechnol. 35B, 198204. 27 Younes, M., Wagner, H. and Strubelt, O. (1989) Biochem. Pharmacol. 38, 3573-3581.
28 Terrier, F., Vock, P., ('otting, J., Ladebeck, R., Rcichcn. 1 and HentscheI, D.(1989) Radiology 171, 557 5(~3. 29 Sakr, M.F.. Zelti. G.M., Hassnein, T.I., Farghali, tt., Nalcsnik, M.A., Gavaler, J.S., Starzl. T.E. and Van Thiel. I).11. (19911 Hepatology 13, 947 951. 30 Brindle, K. and Krikler, S. (1985) Biochim. Biophys. Acla 847, 285-292. 31 Santos, H. and Turner, D.L (1986)J. Magn. Reson. (3S, 345-349. 32 Voci, A., Gallo, G., Balestero, F. and Fugassa, F]. (19881 ('ell Biol. Int. Rpts. 12. 647-660. 33 Goldstein, D.B. (1983) Pharmacology of Alcohol, Oxford University Press, New York. 34 Hernandez-Munoz, R., Santamaria, A.. Garcia-Sainz. J.A., Pina, E. and De Sanchez, C. (1978) Arch. Biochem. Biophys. 190, 155-162. 35 Gillam, E. and Ward, L.C. (1986) Int. J. Biochem. 18, 1031-1038. 36 French, S.W. (1966) Proc. Soc. Exp. Biol. Med. 121,681-689. 37 Baranyai, J.M. and Blum, J.J. (1989) Biochem. J. 258, 121-140. 38 Thurman, R.G. and Scholz, R. (1977) Eur. J. Biochem. 75, 13-21. 39 Yuki, T., Thurman, R.G., Schwabe. U. and Scholz. R. (1980) Biochem. J. 186, 997 1000. 40 Yuki, T., Israel, Y. and Thurman, R.G. (1982) Biochem. Pharmacol. 31, 2403-24(17. 41 Stowell, K.M. and Crow, K.E. (1985) Biochem. J. 262, 595-6(12. 42 Takahashi, H., Geoffrion, Y., Butler, K.W. and French, S.W. (1990) Hepatotogy I 1, 65 73.