In vivo proton nuclear magnetic resonance imaging and spectroscopy studies of halocarbon-induced liver damage

MAGNETIC RESONANCE IN MEDICINE 9,229-239 (1989)

In Vivo Proton Nuclear Magnetic Resonance Imaging and Spectroscopy Studies of Halocarbon-Induced Liver Damage

MANFRED B R A U E R , * ~ ~ RHEAL A. TOWNER,* IAN RENAUD,* EDWARD G. JANZEN,*’t A N D DAVID L. FOXALL$

*Guelph- Waterloo Center for Graduate Work in Chemistry, University of Guelph, Guelph, Ontario, Canada NI G 2 WI, and $Spectroscopy Imaging Systems, 1120 Auburn Street, Fremont, California 94538

Received April 27, 1988; revised July 18, 1988

Proton magnetic resonance imaging and localized NMR spectroscopy were used to study the rat liver in situ. Respiratory gating was used in both the imaging and the localized spectroscopy studies to control for the movement of the upper abdomen of the rat during breathing. After administration of carbon tetrachloride, bromotrichloromethane, or halothane, localized regions of high proton signal intensity were observed in the NMR images of the liver. Localized (VOSY) proton NMR spectra from within these regions indicated that the increase in a signal intensity was due to a longer Tz relaxation time for the water resonance, indicating acute edema in the region of tissue damage. o 1989 Aca-

demic Press, Inc.

INTRODUCTION

Halocarbons, such as carbon tetrachloride ( CCl, ) and bromotrichloromethane ( BrCC13), represent an intensively studied family of classic hepatotoxins. Their pri- mary mechanism of toxic action occurs after they are metabolized in the liver by the cytochrome P-450 system to a variety of free-radical intermediates. For example, CC14 is metabolized in the endoplasmic reticulum of hepatocytes to trichloromethyl radical (. CC13) which, in the presence of oxygen, is thought to be converted to the corresponding peroxy radical (. 0OCCl3) ( 1 , 2 ) . It is generally accepted that it is the trichloromethyl peroxy radical that initiates a degradative lipid peroxidation chain reaction with susceptible unsaturated bonds of the fatty acyl moieties within the phos- pholipids of the hepatocyte membranes. The free-radical mediated attack on the membranes results in increased cellular permeability to extracellular ions and water with eventual cell necrosis and cell death.

Magnetic resonance imaging (MRI) techniques have not been used as extensively on the liver as in other organs, partly because of the problem of respiratory motion artifacts (3-5). The application of MRI specifically to halocarbon-induced hepatotoxicity has been very limited indeed. Stark and co-workers working on a 0.35-T NMR imaging system have studied the effects of various hepatotoxins, including ethionine, iron, and CCl, on the livers of rats (6) to determine whether fatty infiltration,

t To whom correspondence should be addressed at the Magnetic Resonance Imaging Facility of the University of Guelph, 1601 Biomedical Sciences Bldg., OVC, Guelph, Ontario, Canada NIG 2W1. This is publication 001 supported by the University of Guelph MRI Facility.

0740-3194/89 $3.00 Copyright 0 1989 by Academic Press, Inc All rights of reproduction in any form reserved.

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230 BRAUER ET AL.

hepatitis, and fibrosis can be detected by MRI. They found that the intensity of the proton signal increased with CC1, treatment, as did both T , and T2 values for the proton resonances within the liver. Other workers ( 7) however have reported that no appreciable change in the relaxation times of the rabbit liver proton resonances at 0.20 T could be found.

We have investigated CC14 -induced hepatotoxicity at 4.7 T using respiratory gating to obtain images of better resolution. In addition, we have employed localized proton spectroscopic methods to determine the contribution of water and fat toward the overall proton signal and to the observed overall proton relaxation times.

METHODS AND MATERIALS

CCI4 and BrCCl, were obtained from Aldrich Chemical Co. Wistar rats (200-250 g) were given two doses of CC14 or BrCC13 separated by 1 h (1 60 PI/ kg body wt, ip). After the second administration of halocarbon, the rat was anesthetized with sodium pentobarbital (Somnotol 1 m1/2.27 kg body wt, ip). Halothane was administered as a gas for the halothane treatment experiment. Five percent halothane was used to induce anesthesia and 1 % levels were used to maintain the anesthesia.

The anesthetized rat was placed in a special restraining cage specifically designed in conjunction with a respiratory gating device (Fig. 1A). A small balloon filled with deuterium oxide was placed under the rat. The balloon was connected to a length of tubing which contained a column of deuterium oxide. The inspiration of the rat caused an increase in pressure within the balloon which raised the column of liquid. The pressure was then transferred via an increase in air pressure within the tubing to a Honeywell Microswitch ( 142 PCO 1 D) pressure transducer (Fig. 1 B) . This increase in pressure during inspiration was used to trigger acquisition of phase-encoding steps in the imaging sequence.

All the NMR studies described in this study were performed on a Spectroscopy Imaging System 200/ 330 spectrometer at a magnetic field strength of 4.7 T and a 33- cm horizontal-bore magnet. Multiple slices were taken in the transverse plane using a TE of 18 ms. The image slices were 3 mm thick; field of view was 6 X 6 cm, with 256 phase-encoding steps, two acquisitions per step, and 5 12 frequency-encoding points yielding a 256 X 256 pixel image.

Localized proton spectra were taken from ( 4-mm)3 voxels within the liver based on spatial coordinates determined from the images. The VOSY (volume selective spectroscopy) method, based on stimulated echoes methods (8, 9 ) , was used for spatial localization of the proton spectra. A 12-ms delay was used after the first and third 90" pulses, and a 65-ms delay was used after the second 90" pulse. As with the imaging protocol, the pulse sequence was gated with the respiration of the animal.

Localized T2 determinations were obtained for proton resonances within the voxels by a modification of the VOSY method. The delay after the first and third 90" pulses was incremented from 12 to 40 ms in 2-ms increments, while the middle delay was kept constant at 65 ms. T2 determinations were calculated via nonlinear regression analysis.

RESULTS

Control rats were imaged after they had been anesthetized with pentobarbital. Sag- ittal images were initially taken to establish the coordinates for multiple transverse

'H NMR IMAGING OF HALOCARBON-DAMAGED LIVER 23 1

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FIG. 1 . (A) Physical layout of the respiratory gating device and restraining cage. In order to gate an image acquisition to the expiration of an anesthetized rat the animal was placed within a polycarbonate restraining cage ( A ) with its abdomen placed over a balloon filled with deuterium oxide (1). A column of the deuterium oxide also partially filled a manometer (2) and the column of fluid moved up and down with the respiration of the rat. The rat was held in place by an adjustable restraint (3). The pressure rise and fall due to the rat's breathing was transmitted through the air within a long length of tubing out of the magnet to a pressure sensor in the gating device (B ) . A light indicator (4) would flash when a gating impulse was sent to the external trigger input of the spectrometer. The sensitivity of the device was adjustable ( 5 ) . (B) Schematic diagram of the respiratory gating device.

slices. The position of the liver was readily apparent in the sagittal images as a fairly homogeneous region between the dark outline of the lung and the relatively high- intense region ofthe viscera. Transverse images taken through the liver region showed a lack of anatomical detail due to the effect of respiratory motion (images not shown). Images in which the pulse sequence was gated to the expiration of the rat showed much greater detail (Fig. 2A). A number of major blood vessels are readily discernible with respiratory gating, which could not be resolved from surrounding

FIG. 2. (A) Normal rat liver. A respiratory gated magnetic resonance image was obtained as a transverse slice through the liver of a normal rat. The stomach is clearly visible as a region of lower intensity in the upper left part of the image and the spinal chord is a bright region near the top of the image. The liver is the large, relatively homogeneous region below and to the right of the stomach. (B) CCL-treated rat liver. An analogous image was obtained 3 h after CCL treatment. Note the area of high signal intensity in the liver to the right of and slightly below the stomach.

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'H NMR IMAGING OF HALOCARBON-DAMAGED LIVER 233

tissue in the absence of gating. All subsequent images were gated to the respiration of the rat.

Since each acquisition was gated to one expiration, the repetition time (TR) was controlled by the rat. Typically, the rat breathed once every 0.8 to 1.4 s. The echo time (TE) was kept fairly short at 18 ms, as the signal intensity of the liver decreased substantially at longer TE times, indicative of a short liver T, value.

CC4-treated rats were given two ip doses 1 h apart, anesthetized after the second dose, and imaged. Transverse images taken 2-4 h after the second dose of CCL, showed a region of the liver with dramatically increased proton signal intensity (Fig. 2B) compared to the control rat (Fig. 2A). This region of the liver corresponds to the location where the portal vein initially enters the liver. Other regions of the liver did not change signal intensity in the image. Transverse images taken 16 h after the CC14 treatment did not show as dramatic an effect as the 2- to 4-h images. However, after I 6 h, an appreciable accumulation of lower peritoneal fluid was present in the rats and was detectable upon palpation or as intense regions of proton intensity in transverse images through the lower abdomen.

BrCC13, a more potent hepatotoxin, was administered in the same way as CC14, and again images were taken following the second dose of toxin. Very similar effects were seen in the transverse slices at the level of the entry of the portal vein into the liver (Figs. 3A and 3B). The major difference was that the region of increased signal intensity developed after only 1 h. Again, this increased intensity had subsided after I 6 h and an increase in peritoneal fluid could be detected.

Halothane (CF,CHClBr ) is a commonly used gaseous anesthetic used clinically for major surgical procedures. It is a halocarbon chemically similar to CCL, or BrCCl,. Similar to CC14, a significant portion of halothane undergoes reductive metabolism via cytochrorne P-450, to 2-chloro- 1, 1, 1-trifluoroethyl radical, and subse- quently to other toxic free-radical intermediates (10). Halothane is known to cause hepatic damage, inflammation, and hepatocyte swelling in rats under conditions similar to those of our experiments ( 11 ) and can cause liver damage clinically under certain conditions (12) . When a rat was anesthetized with halothane for 4 h and transverse images were taken through the liver, the same region of the liver showed a significant increase in signal intensity (Figs. 4A and 4B).

In order to establish whether the increase in signal intensity in the rat liver caused by CCb, BrCCl,, or halothane is due to an increase in the water resonance or the fat signal (methyl and methylene protons of fatty acyl moieties), localized proton spectroscopy was attempted. The VOSY method was used, with the spatial coordinates determined from the transverse images. Spectra taken in regions of apparent tissue damage showed a very large water proton resonance compared to the regions of the liver apparently unaffected by the hepatotoxin (Figs. 5A and 5B). As with the imaging experiments, data acquisition was triggered by the respiration of the rat. The shoulders on the water proton resonance are due to residual eddy current effects following the last selective 90" pulse. This effect also contributes to the observed line- widths.

The VOSY method was modified so as to obtain relative T2 values within selected voxels of the rat livers. The regions of apparent liver damage showed significantly longer water proton T2 values than regions of unaffected liver (Figs. 6A and 63) . Apparent proton T2 values in regions of increased signal intensity ranged from 20 to

234 BRAUER ET AL.

FIG. 3. (A ) Normal rat liver. See Fig. 2A. (B) BrCC13-treated rat liver. Transverse slice through the liver 2 h after BrCC13 treatment. Note the area of high signal intensity to the right of the stomach, as in Fig. 2B.


FIG. 4. (A) Normal rat liver. See Fig. 2A. (B) Halothane-treated rat liver. Transverse slice through the liver 4 h after initiation of 1% gaseous halothane. Again note the increases signal intensity to the right of and slightly below the stomach. (The stomach also has some high signal intensity due to the presence of food).

236 BRAUER ET AL.

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FIG. 5. (A) In vivo proton NMR spectrum of an unaffected region of liver. Localization was achieved by the VOSY method (see Methods and Materials). The predominant resonance ( 6 N 4.5 ppm) is that of water with negligible peak intensity for the methylene protons of fat ( 6 Y 1.0 ppm). (B) In vivo proton NMR spectrum of an injured region of liver. Localization was achieved by the VOSY method. Halothane was used to induce tissue injury and the region from which this spectrum was taken corresponds to a (4- mm)3 voxel in the center ofthe region of high signal intensity in Fig. 4B.

30 ms, while unaffected regions of the liver had apparent T2 values of 10 ms. Liver from control rats also had apparent T, values of approximately 10 ms. No significant deviation of the data from a monoexponential decay was observed during the time measured for either the affected or the unaffected regions of the liver.

DISCUSSION

By modification of the VOSY method, an apparent T2 for the proton resonance of water within the liver was determined to be 10 ms at 200 MHz. The proton T2 values determined by the modified VOSY method were uncorrected for the effects of molecular diffusion. However, relative changes in T2 between voxels of healthy and damaged liver do reflect real changes in the relaxation rates due to tissue injury. Literature values for in vivo normal liver are reported as 37 ms at 15 MHz (6) and 30 ms at 30 MHz ( 13) . These values are for both water and fat protons and employ the two-point method of T2 determination. No literature values for the water resonance alone have been reported for in vivo rat liver at 200 MHz.

The increase in observed signal intensity observed in the region of the liver where the portal vein enters the organ is consistent with the proposed mechanism of halocarbon-induced hepatotoxicity. CC14 and BrCC13 when given intraperitoneally are


FIG. 6. (A) T2 relaxation decay in region of normal liver. The T2 relaxation time was determined using a modification of the VOSY method (see Methods and Materials). The spectra correspond to Fig. 5A. (B) T2 relaxation decay in region of injured liver. The spectra correspond to Fig. 5B.

brought to the liver directly through the portal vein. The toxic effects of these com- pounds may require that they be first metabolized to free-radical intermediates in the liver. Halothane also induces an increase in signal intensity in the same region of the liver. It is administered to the rat as a gas and would thus enter the liver through either the portal vein or the hepatic artery. Since the hepatic artery also enters the liver in the same region as the portal vein, it is not too surprising that the same effect was observed in a similar region of the liver.

The increase in observed proton signal due to halocarbon-induced hepatotoxicity could be attributed to a variety of possible causes. One possibility is the induction of local fatty infiltration. Fatty infiltration is a commonly observed effect of hepatotoxins, because of the liver's major role in processing and distribution of triglycerides. The accumulation of fat in the liver of a rat following treatment with ethionine or ethanol has been reported using proton chemical-shift imaging (14). However, our localized proton spectroscopic results indicate that this is not a major mechanism in the acute phase of halocarbon-induced toxicity, since no major increase in the methylene region ( 1 .O to 1.5 ppm) corresponding to fatty acyl protons could be detected. The increased intensity of the water resonance is, however, quite apparent.

The cause for the increased signal intensity (1) after halocarbon administration in both the images and in the VOSY spectra is due to a change in the water resonance. Four characteristics of the water resonance may alter the observed intensity: d, the density of protons; the T , and the T2 for the proton resonance; andf( u ) , a function of the flow of the water,

Z = d X f ( v ) X exp( -TE/ T,) X [ 1 - exp( -TR/ T, ) ] .

An increase in the amount of water present in the liver is possible. Since the normal liver is already about 68% water and the water content typically rises by less than 10%

238 BRAUER ET AL.

for edematous tissue ( 1 5 ) , an increase in water content per se would not be sufficient in itself to explain the dramatic (greater than threefold) increase in proton signal intensity observed. Thef( v) factor is important for the flow of blood through blood vessels, but is not relevant for the stationary regions of the liver itself.

The increase in signal intensity was not due to TI effects because of the slow repetition times (TR) for data acquisition compared to the known T I relaxation times for liver proton signals. Typical literature values for T, of liver in vitro and in vivo range from 240 to 400 ms for 15 to 100 MHz frequency (6, 15) . If we assume a TI of 400 ms at 200 MHz proton frequency and a typical TR (based on the respiration rate of the rat) of 1000 ms, the steady-state signal amplitude observed should be 0.92 of the absolute amplitude at infinite TR. Hence the images are not appreciably TI weighted.

The major mechanism for increased proton signal intensity is a dramatic increase in the TI values of the water resonance in the damaged areas of the liver. An increase in T2 from 10 to 30 ms would produce an increase in signal intensity of 3.3-fold for an imaging sequence with TE of 18 ms. Thus, the increase in T2 induced by halocarbon administration can account for the dramatic increase in observed proton signal intensity.

The increase in water T2 is likely due to local edema as a result of the halocarbon- induced tissue damage. The T2 values of water resonances are strongly dependent upon the amount of “bound” water present in tissue (13, 16) . The overall observed water T2 value ( TZbs) is a fast-exchange averaged value determined from the T2 of “free” water ( T r ) , the very short T2 of water bound to proteins and membranes ( T?) , and the fraction of total water in the free (Ff) and bound (1 - Ff) pools: 1 / Tqbs = Ff/ T: + (1 - Ff)/ T:. Local tissue damage caused by the metabolism of CCL, BrCC13, or halothane could induce an inflammatory response resulting in, among other things, edema. This localized edematous response increases the molar fraction of “free” water and hence decreasing the molar fraction of “bound” water. Since the observed T2 is dominated by the (1 - F‘)/ TF term, a small decrease in the fraction of “bound” water will result in a large increase in observed water T2. The loss in this increased proton signal intensity after 16 h is likely due to resorption of this excess free water as the tissue repairs itself or drainage of the extracellular water into the peritoneal cavity.

In summary, these studies demonstrate the sensitivity of NMR imaging and localized spectroscopy in studying the response of in vivo biological systems to toxicologi- cal challenge.

ACKNOWLEDGMENTS

We thank Dr. Peter S. Allen (Alberta Heritage Foundation for Medical Research, University of Alberta, Edmonton, Alberta, Canada) for his helpful suggestions for the respiratory gating device. We also thank Casey Gielen (Machine Shop, Physics Department, University of Guelph) for his assistance in the design and construction of the restraining apparatus. Financial support was provided by the University ofGuelph MRI Facility.

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In vivo proton nuclear magnetic resonance imaging and spectroscopy studies of halocarbon-induced liver damage