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Effect of Experimental Hepatic Injury on in vitro Drug-Metabolizing Enzyme Activities in the Rat
Vol. 73, No. 4, Part 1
GASTROENTEROLOGY 73:691-696, 1977 Copyright © 1977 by the American Gastroenterological Association
Printed in U.SA .
EFFECT OF EXPERIMENTAL HEPATIC INJURY ON IN VITRO DRUGMETABOLIZING ENZYME ACTIVITIES IN THE RAT R. A.
WILLSON,
M.D.,
AND
F. E.
HART,
B.S.
Division of Gastroenterology, University of Washington , Seattle, Washington
Experimental hepatic necrosis was induced in phenobarbital pretreated rats by means of the intraperitoneal administration of an acetaminophen-dimethyl sulfoxide mixture over a dosage range of 0.3 to 1.5 g per kg. At lower doses, cytochrome P-450 concentration and the specific activities of aminopyrine demethylase and bilirubin glucuronyl transferase increased, whereas aniline hydroxylase activity was mildly decreased. In contrast, &t higher doses, cytochrome P-450 concentration and the specific activities of all of the enzymes were decreased significantly (P < 0.01), as compared with control rats. The diminished activities, however, were generally modest, not uniform for all enzymes assayed, and correlated poorly with histological necrosis and routine laboratory tests. When necrosis was severe, a prominent peak was detected at 420 nm, and this was associated with a more consequential decrease in all of the enzyme activities. When the total in vitro hepatic drug-metabolizing enzyme capacity was calculated, only the cytochrome P-450 content and aminopyrine demethylase activity were significantly decreased at the 1.5-g per kg dose level. It is concluded that in acute liver disease changes in the hepatic in vitro drug-metabolizing enzyme capacity may not be closely related to cellular necrosis, per se, and the degree of change in enzyme activities will vary from one enzyme system to another. These findings may explain, in part, the often inconsistent alterations in the disposition and elimination of drugs described in associated liver diseases. The disposition of drugs, whose major biotransformation is within the. liver, may be significantly altered with associated hepatic disease; however, some drugs may not be affected, and the degree of alteration may be variable. 2 Two recognized factors that may in part account for these inconsistencies are the complex metabolic pathways involved in drug biotransformation, and the heterogeneity of the patients studied as regards stage or severity of liver disease. In acute hepatic injury, a reduced drug clearance may be related to a number of pharmacokinetic factors, however, an impaired extraction by the liver may be a principal cause. 3 Both diminished uptake and/or rate of metabolism would result in a reduced drug extraction ratio. Concerning the rate of metabolism, some in vitro drugmetabolizing enzyme activities have been assayed on human liver biopsy specimens, however, their activities
were not greatly reduced unless the extent of necrosis was severe,4 and not all enzyme activities were uniformly affected. Further, a poor correlation between in vitro drug-metabolizing enzyme activities and routine "liver function" tests has been reported in acute hepatitis, both in the initial episode and during resolution. 5 Because of these limited observations, it was thought that a systematic evaluation of some selected in vitro drug-metabolizing enzyme activities under uniform experimental conditions might help to clarify the relative role of the in vitro drug-metabolizing enzyme capacity in altered drug disposition when associated with acute liver injury. Consequently, the purpose of the present study was to investigate the relationship between in vitro drug-metabolizing enzyme activities, liver necrosis, and routine laboratory tests in the rat with acetaminophen-induced hepatic injury.
Received July 6, 1976. Accepted April 14, 1977. Materials and Methods This paper was presented in part at the Annual Meeting of the Male Sprague-Dawley rats (240 to 260 g) were used in all American Association for the Study of Liver Diseases, Chicago, Illinois, November 4 and 5, 1975, and has been published in abstract experiments. They had free access to food (Purina rat chow, Ralston Purina Company; St. Louis, Mo.) and water, and were form.' Address requests for reprints to: Richard A. Willson, M.D., Har- caged in a constant temperature room (22°C) with alternating borview Medica l Center, 325 9th Avenue, Seattle, Washing- 12 hr of light and darkness. All studies were done in the early morning. ton 98104. This study was supported in part by Grant AM 17813 from the Preparation of the hepatotoxin. Acetaminophen (4'hydroxyUnited States Public Health Service. acetanilide, Eastman Organic Chemicals, Rochester, N.Y.), a Dr. Willson is a recipient of Academic Career Development known liver toxin, ~ was assessed for purity by thin layer Award AM-00151 from the National Institute of Arthritis, Metabo- chromatography on Silica Gel G in ethyl acetate-methanollism and Digestive Diseases. water-acetic acid, 60:30:9:1, v/v. 7 In our experience, the dos691
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WILLSON AND HART
ages of acetaminophen that we planned to utilize were not consistently soluble in warm saline at alkaline pH. 8 Consequently, 0.5 ml of dimethyl sulfoxide (DMSO) (Fisher Scientific Company, Fairlawn, N.J., spectral grade) was used as a solvent in the preparation of the acetaminophen for intraperitoneal administration. The DMSO was chromatographed in the same system and had an R distinct from that of acetaminophen. The acetaminophen-DMSO mixture was assessed chromatographically also and produced R values corresponding to their respective components with no evidence for a drugDMSO complex, as has been reported for some compounds. 9 To investigate further the possibility of drug interaction with DMSO, gas liquid chromatography-mass spectroscopy (kindly performed by Dr. V. Raisys, Associate Director, Toxicology Laboratory, Harborview Medical Center, Seattle, Washington) was performed on the mixtures. The sensitivity of this analysis was such that it would detect the mixture down to 100-ng quantities. Again, each compound was pure and there was no evidence for drug-DMSO interaction. Animal preparation. Because microsomal enzyme induction enhances the h epatic necrosis caused by acetaminophen, 8 all rats were pretreated with phenobarbital (75 mg per kg per day) intraperitoneally for 4 days before the administration of the acetaminophen. Then the rats were divided randomly into two groups. The first was given 0.5 ml of DMSO intraperitoneally and are considered as the treatment controls. The second received the acetaminophen mixture intraperitoneally over a dosage range of 0.3 to 1.5 g per kg and are considered as the treatment group. Preparation of tissue fractions. The rats were fasted overnight and killed by decapitation approximately 72 hr after the acetaminophen administration. Mter decapitation, the liver was perfused in situ with 75 ml of ice-cold 0.15 M sodium chloride, and then excised, weighed, and biopsied. One gram of t he liver was then homogenized with 9 ml of0.25 M sucrose in 0.001 M ethylenediaminetetraacetate (EDTA),pH 7.4, with a glass pestle and homogenizing tube. Six grams of the remaining liver were homogenized with 12 ml of 0.01 M sodiumpotassium phosphate with 1.15% potassium chloride, pH 7.4. Microsomes from both homogenates were prepared by differential centrifugation at 105,000 x g for 1 hr in a Beckman L265 B ultracentrifuge (Beckman Instruments, Inc. , Fullerton, Calif.). The microsomal pellets resulting from the sucrose homogenates were resuspended in 5 ml of 0.25 M sucroseEDTA and used for the glucuronyl transferase assay. The pellets resulting from the sodium-potassium phosphate homogenates were resuspended in 10 ml of 0.05 M sodium-potassium phosphate and were used for the aminopyrine demethylase, aniline hydroxylase, and cytochrome P-450 assays. Enzyme assay. Protein concentrations of all microsomal suspensions were determined by the method of Lowry et al. 10 using bovine serum albumin as standard. Concentrations of protein were adjusted for optimal conditions. Repeat Lowry determinations were done on the adjusted samples. Incubations were carried out in a 37oC shaking water bath for all assays. Aminopyrine demethylase activity was assayed according to the method given by Mazel 11 with the following modifications in order to give maximal activity: 20 p.moles of aminopyrine, 2.5 units of glucose-6-phosphate dehydrogenase, and 5 mg of microsomal protein were added to the incubation. The formaldehyde produced was estimated by the method of Nash. 12 Aniline hydroxylase activity was assayed according to the method given by Mazel 13 with the following modifications in order to give maximal activity: 20 p.moles of aniline-HCl, 1 p.mole ofNADP, 2.5 units ofglucose-6-phosphate dehydrogenase, and 10 mg of microsomal protein were added to the incubation mixture. Bilirubin glucuronyl transferase activity
Vol. 73, No.4, Part 1
was assayed utilizing the method of Black et al. 14 Cytochrome P-450 content was measured by the method of Omura and Sato 15 using a Beckman 25 spectrophotometer in the sp lit beam mode. Liver histology . Initially, liver specimens were taken from four arbitrary areas in the liver and evaluated for their uniformity in the representation of necrosis. Because there was little difference among the four areas sampled, only one section was taken for the remainder of the study. The specimens were fixed in formalin and stained with hematoxylin and eosin. The sections were graded by light microscopy into minor necrosis (grade I), moderate necrosis (grade II), and severe necrosis (grade III).'" Biochemical indicators of liver injury. Serum bilirubin concentrations were assessed spectrophotometrically as described by Malloy and Evelyn. 17 Serum glutamic oxaletic transaminase (SGOT) activities were assayed by the colorimetric method of Sigma at 505 nm (Sigma Chemical Company, St. Louis, Mo.). Blood samples were drawn for these analyses before treatment and at the time of death. Expression of data. Microsomal drug-metabolizing enzyme specific activities are expressed as product formed per unit time per milligram of microsomal protein, and as an estimate of a hepatic drug-metabolizing enzyme capacity by the product formed per unit time per total liver weight per 100 g of body weight. Analysis of data . Statistical indices were calculated by standard methods and are expressed as the mean ± SEM. A ttest of the difference between two sample means was utilized to assess significance between parameters. 18
Results and Discussion Effect of acetaminophen dosage upon mortality and hepatic necrosis. Table 1 illustrates the relationship between acetaminophen dosage, hepatic necrosis, and mortality. At the 300-mg per kg dose schedule·there was neither hepatic necrosis nor rat death. Both mortality and the percentage of rats with histological necrosis showed a commensurate increase with dose up to the 1200-mg per kg level. There was, however, considerable variation in the presence and extent of necrosis between rats, and this has been observed by others. 19 At the 1500-
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October 1977
mg per kg dose, the percentage of animals demonstrating histological necrosis decreased, although the mortality continued to increase. This suggested that at the highest dose, death was related to causes other than hepatic necrosis. Such a conclusion was further supported by the fall in serum transaminase at this dose schedule (fig. 2). Complete autopsies were not done on the rats so other organ damage is only postulated. A previous report also noted a similar discordance between mortality and hepatic necrosis, and the cause of death was not apparent. 8 Acetaminophen-induced hypoglycemia has been postulated as a cause, 20 but we did not assess blood glucose levels. Death of the rats usually occurred within 12 to 24 hr after the acetaminophen administration. Effect of acetaminophen dosage upon drug-metabolizing enzyme specific activities and hepatic drug metabolizing enzyme capacity. All acetaminophen-treated rats lost weight as the dosage schedule increased, and at the higher doses the liver weight increased. The calculated gram liver weight per 100 gram of body weight (mean ± SEM) was as follows: control (3.51 ± 0.25), 300 mg per kg (3.45 ± 0.17), 600 mg per kg (3.92 ± 0.35) 900 mg per kg (3.68 ± 0.35), 1200 mg per kg (4.24 ± 0.50), 1500 mg per kg (4.28 ± 0.58). Table 2 shows the change in microsomal protein concentrations and the enzyme-specific activities over the corresponding dose schedule. The microsomal protein concentration represents our yield following ultra centrifugation. There was a trend for the enzyme activities to increase over the lower doses (300 and 600 mg per kg), however, these changes were not significant. As the acetaminophen dose schedule increased, the cytochrome P-450 concentration and the aminopyrine demethylase activity decreased, and at the 1500-mg per kg dose, both were approximately 60% of control value. In contrast, the aniline hydroxylase and TABLE 1. Effect of acetaminophen dosage upon hepatic necrosis and mortality in rats Rats with histologiMortality Acetaminophen dose cal necrosis mg/kg % % 300 (6)" 0 0 600 (14) 21 0 900 (22) 30 9 1200 (29) 40 31 1500 (27) 31 52 a
Number of animals in parentheses.
Treatment mg!kg
Control 300 600 900 1200 1500
glucuronyl transferase activities were not as affected at the 1500-mg per kg dose level, both being about 80% of control value. Therefore, all enzyme specific activities were not decreased equally, and the diminished activities, although significant, were generally modest. The estimation of the hepatic drug-metabolizing enzyme capacity (table 3) revealed a similar trend for the enzyme activities to increase at the lower dosage levels, and at the 1500-mg per kg dose, only the cytochrome P-450 and aminopyrine demethylase were significantly decreased. The nonuniformity in the decrease of enzyme activities has previously been reported. Certain hydroxylase enzymes are less affected by experimental necrosis, 21 and this may be related to the regionalization of drug-metabolizing enzyme activities within the hepatic lobule. 22 Further, experimental cholestasis studies have shown that the decrease in aniline hydroxylase activity was less than the aminopyrine demethylase activity, and this was related to the involvement of different binding sites on the cytochrome P-450 molecule. 23 Lastly, it has recently been reported that the glucuronidation pathway in drug disposition may not be as affected by liver injury as the biotransformation pathways via the mixed function oxygenase system. 24 At the 900-, 1200-, and 1500-mg dose levels, there was a prominent spectral peak at 420 nm (fig. 1) noted in 16,
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FIG. 2. The changes in serum transaminase (SGOT) over the acetaminophen dose schedule. Ll.SGOT represents the mean difference between pretreatment and post-treatment (72 hr) values for SGOT in the same animals. The number of animals at each dose is the same as that given in table 2.
TABLE 2. Effect of acetaminophen dose on drug-metabolizing Aminopyrine demethylMicrosomal protein Cytochrome P-450 ase HCHO formed!mg nmoles mg/g liver wt nmoles!mg protein protein/min 36.82 ± 1. 73 (33) 1.70 ± 0.07 (28) 5.45 ± 0.43 (31) 43.10 ± 3.33 (4) 1.93 ± 0.11 (4) 6.29 ± 0.16 (4) 45.93 ± 2.54b (14) 1.81 ± 0.09 (8) 5.97 ± 0.50 (14) 34.76 ± 1.28 (19) 1.26 ± 0.1~ (15) 4.55 ± 0.56 (15) 37.15 ± 1.37 (19) 1.21 ± 0.08b (19) 4.09 ± 0.35C (18) 33.16 ± 1.41 (13) 1.05 ± 0.07b (13) 3.36 ± 0.34b (13)
Values given are the mean± SEM with number of observations in parentheses. P < 0.01 as compared with control. c p < 0.05 as compared with control. a
b
693
DRUG-METABOLIZING ENZYMES IN HEPATIC INJURY
enzyme activities•
Aniline hydroxylase
Glucuronyl transferase
nmoles p-aminophenol formed/mg protein/min 1.05 ± 0.02 (27) 1.00 ± 0.02 (4) 0.89 ± 0.03 (4) 0.90 ± 0.06b (19) 0.97 ± 0.06 (19) 0.87 ± 0.06b (13)
pg bilirubin formed!mg protein/min 1.29 ± 0.04 (28) 1.51 ± 0.09 (4) 1.51 ± 0.09 (10) 0.95 ± 0.08b (14) 1.10 ± o.w (18) 1.02 ± 0.07b (13)
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WILLSON AND HART
16, and 25% of the rats, respectively. This spectral peak was associated with grade II and grade III hepatic necrosis, and · a more consequential decrease of the hepatic drug-metabolizing enzyme capacity as compared with their mean levels for these three dose schedules (table 4). For example, at the 900-mg per kg dose level, the cytochrome P-450 content and the activities of aminopyrine demethylase, aniline hydroxylase and glucuronyl transferase were 36, 47, 46, and 46% of their control values, respectively. Therefore, with severe necrosis all enzyme activities were similarly decreased. The alteration of cytochrome P-450 to a probable P-420 degradation product has been reported previously. 25• 26 The relationship between drug-metabolizing enzyme activities and hepatic necrosis. Although the aminopyrine demethylase activity and cytochrome P-450 concentration diminished with increasing doses of acetaminophen, their decrease did not correlate well with the estimated extent of necrosis. At the 900-, 1200-, and 1500-mg dose levels, the correlation between cytochrome P-450 and hepatic necrosis was -0.77, -0.56, and -0.55, respectively; similar values were found with the aminopyrine demethylase activity. This rather poor correlation with cellular necrosis could, in part, reflect the probable inaccuracy of the qualitative assessment of the tissue injury. Utilizing several techniques for assessing this, Dixon found considerable variation in the extent of necrosis from different parts of the rat liver, even with multiple tissue blocksY We observed that the extent of hepatic necrosis when present was generally modest and this is in agreement with a previous report over a similar dose range. 8 The route of administration may be important as regards development of hepatic necrosis, in that oral acetaminophen19• 27 appears to induce hepatic necrosis more readily than does the intraperitoneal route, 8 however, the dose administered orally TABLE
Treatment
3. Effect of acetaminophen dose on drug-metabolizing enzyme capacity of the liver'
Cytochrome P-450 nmoles/totalliver wt/100 g body wt
mg/kg
Control 300 600 900 1200 1500
216.23 282.67 328.58 150.92 190.23 148.27
is generally larger. An additional explanation could be that the biotransformation of drugs within the enterocyte of the proximal small intestine28• 29 may play a role in drug-induced liver injury, similar to the now recognized role of hepatic drug biotransformation. 30 The rat is recognized to be more resistant to acetaminophen than other species3 1 and to have a variable hepatic injury response. 8 In addition, we have reported that DMSO diminishes the activity of the mixed function oxygenase enzyme system in the phenobarbital pretreated rat,32 and this reduced activity would lessen the toxicity of acetaminophen. Alternatively, previous studies suggest that perhaps a poor relationship with necrosis should not be unexpected, as some drug-metabolizing enzyme activities are diminished in cholestasis, 23 where hepatocellular necrosis is not prominent; some in vitro enzyme activities are decreased after CC1 4 administration without cellular necrosis; 3 and some in vitro enzyme activities are not decreased even when hepatic necrosis is quite marked.4 Finally, in a 3-day sequential study, we found that all enzyme specific activities were diminished by 24 hr without evidence for histological necrosis. 34 The relationship between drug-metabolizing enzyme activities and routine laboratory tests . Figure 2 illustrates the changes in serum oxalacetic transaminase levels over the acetaminophen dose range, with the greatest elevation being at the 1200-mg per kg dose, followed by a decrease at the 1500-mg dose level. Therefore, at the highest dose, the SGOT elevation was minid mal, whereas both the cytochrome P-450 concentration and the aminopyrine demethylase activity were at their lowest levels. It is apparent then that the relationship was limited. The changes in serum transaminase, however, did reflect hepatic necrosisn as the correlation coefficient between the changes in transaminase and
Aniline hydroxylase Aminopyrine demethylase nmoles HCHO formed/min/total liver nmoles p-aminophenol formed/ wt/100 g body wt min/total liver wt/100 g body wt
± 85.80 (28) ± 23.18 (4) ± 92.40 (8)
728.87 889.58 1041.14 589.05 643.33 447.22
± 56.54b (15) ± 63.30 (19) ± 40.45C (13)
68.18 (31) 151.59 (4) 109.57" (14) 69.30 (15) 64.32 (18) ± 35.13" (13) ± ± ± ± ±
131.43 154.19 167.45 114.10 152.16 119.95
Glucurony I transferase pg bilirubin formed/min/total liver wt/100 g body wt
111.27 134.48 178.80 86.25 126.18 102.28
7.28 (27) 14.48 (4) 14.41 (4) 8.07 (19) 9.94 (19) ± 8.92 (13) ± ± ± ± ±
± 7.91 (28) ± 15.94 (4)
± 16.75 (10) ± 9.74 (14) ± 6.31 (18) ± 9.60 (13)
• Values given are the mean ± SEM with number of observations in parentheses. P < 0.05 as compared with control. c P < 0.01 as compared with control. b
TABLE
4. Drug-metabolizing enzyme capacities in the animals in which a prominent spectral p eak at 420 nm was detected•
Treatment
Bilirubin
SGOT"
mg/100 ml
Control 900 1200 1500
0.1 0.27 0.17 0.20
8 497 1831 72
Aminopyrine deAniline hydroxylase Glucuroriyl transmethylase ferase . nmoles HCHO formed/ nmoles p-aminophenol pg bilirubin formed/ nm~l~~/to~lJwe~ wtl min/total liver wt/100 formed/min/total liver min/total liver wt/100 g 0 Yw g body wt wt/100 g body wt g body wt Cytochrome P-450
216.23 76.26 118.11 98.94
728.87 346.03 341.05 264.76
131.43 60.65 80.20 71.22
111.27 51.19 90.14 50.38
• The drug-metabolizing enzyme activities are mean values tak en from table 3. Values given represent the mean with number of observations in parentheses. b 6.SGOT represents mean difference between pretreatment and post-treatment (72 hr) values for SGOT in the same animals.
DRUG-METABOLIZING ENZYMES IN HEPATIC INJURY
October 1977
histological necrosis 'Vas r = 0. 74, a value similar to that reported by Dixon et al. 27 The poor relationship between routine laboratory tests and the altered disposition of drugs has been reported by a number of authors35· 36 and only with severe liver disease has a correlation been noted with serum albumin and prothrombin time. 37..:39 The dissociation of these tests and clearance of drugs after clinical recovery from acute hepatic disease has further suggested that routine laboratory tests may bear little association with the liver's capacity to metabolize drugs.40 The relationship of serum bilirubin levels, primarily unconjugated bilirubin, with cytochrome P-450 concentration and glucuronyl transferase activity is illustrated in figure 3. The elevation of serum bilirubin was small, but it did rise with the commensurate fall in glucuronyl transferase activity. It has been proposed that the unconjugated hyperbilirubinemia seen early in clinical acetaminophen overdose may be attributable to a decreased glucuronyl transferase activityY In addition, experimental studies in rats suggest that hepatic glucuronide conjugation is impaired early after acetaminophen overdose. 42 Other factors may be involved, however, as it is recognized that the in vitro assay of glucuronal transferase activity does not always correlate well with serum bilirubin levels. 43 As shown in figure3, the cytochrome P-450 concentration also decreased and the serum bilirubin was elevated in the group of animals that demonstrated the 420 nm spectral peak (table 4). It is possible that this 420 peak represents a degraded cytochrome P-450, and it has been suggested that the degraded hemeprotein would be catabolized via hemoxygenase to unconjugated bilirubin. 44 Consequently, our findings indicate that the unconjugated hyperbilirubinemia seen with severe hepatocellular necrosis could be related to both the increased degradation of cytochrome P-450 and the diminished glucuronyl transferase activity. Application to human disease . The extrapolation of in vitro drug-metabolizing enzyme activities to the whole liver's capacity in vivo is difficult, as the in vitro assays are done under quite artificial circumstances and they, 120
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695
of course, do not reflect alterations in the various pharmacokinetic factors that influence drug disposition in vivo. The results of this study, however, do suggest that in acute liver disease, changes in the hepatic in vitro drug-metabolizing enzyme capacity may not be closely related to cellular necrosis, per se, and the degree of change iri. enzyme a<;:tiyities will vary from one enzyme system to another. These findings may explain, in part, the often inconsistent alterations in disposition and elimination of drugs in associated liver disease. The recent reports utilizing a noninvasive breath analysis method for the assessment of drug disposition in liver disease 45 -47 may provide more direct information on altered in vivo drug metabolism and further should provide useful correlations with in vitro drug-metabolizing enzyme activities. REFERENCES 1. Willson RA, Hart FE: Effect of experimental hepatic injury on microsomal drug metabolizing activity (abstr). Gastroenterology 69:880, 1975 2. Wilkinson GR, Schenker S: Drug disposition and liver disease. Drug Metab Rev 4:139-175, 1975 3. McHorse TS, Wilkinson GR, Johnson RF, et al: Effect of acute viral hepatitis in man on the disposition and elimination of meperidine. Gastroenterology 68:775-780, 1975 4. Schoene B, Fleischmann RA, Remmer H, et al: Determination of drug metabolizing enzymes in needle biopsies of human liver. Eur J Clin Pharmacal 4:65-73, 1972 5. Doshi J, Luisada-Opper A, Leevy CM: Microsomal pentobarbital hydroxylase activity in acute viral hepatitis. Proc Soc Exp Biol Med 140:492-495, 1972 6. Dixon MF, Nimmo J, Prescott LF: Experimental paracetamolinduced hepatic necrosis: a histopathological study. J Pathol 103:225-229, 1971 7. Cummings AJ, King ML, Martin BK: A kinetic study of drug elimination: the excretion of paracetamol and its metabolites in man. Br J Pharmacal Chemother 29:150-157, 1967 8. Mitchell JR, Jollow DJ, Potter WZ, et al: Acetaminophen-induced hepatic necrosis. I. Role of drug metabolism. J Pharmacal Exp Ther 187:185-194, 1973 9. Levine WG: Effect of dimethyl sulfoxide on the hepatic disposition of chemical carcinogens. Ann NY Acad Sci 243:185-193, 1975 10. Lowry OH, Rosebrough NJ, Farr AL, et al: Protein measurement with the Falin phenol reagent. J Biol Chern 193:265-275, 1951 11. Mazel P: Experiments illustrating drug metabolism in vitro. In Fundamentals of Drug Metabolism and Disposition. Edited by BN LaDu, HG Mandel, EL Way. Baltimore, Williams & Wilkins Co, 1971, p 546-550 12. Nash T: The colorimetric estimation of formaldehyde by means of the Hantzsch reaction. Biochem J 55:416-421, 1953 13. Mazel P: Experiments illustrating drug metabolism in vitro. In Fundamentals of Drug Metabolism and Disposition. Edited by BN LaDu, HG Mandel , EL Way. Baltimore, Williams & Wilkins Co, 1971, p 569-572 14. Black M, Billing B, Heirwegh KPM: Determination of bilirubin UDP-glucuronyl transferase activity in needle-biopsy specimens of human liver. Clin Chim Acta 29:27-35, 1970 15. Omura T, Sato R: The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J Biol Chern 239:2370-2378, 1964 16. Chalkley HW: Method for the quantitative morphologic analysis of tissues. J Natl Cancer lnst 4:47-53, 1943 17. Malloy HT, Evelyn K: The determination of bilirubin with the
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photoelectric colorimetric. J Bioi Chern 119:481-490, 1937 33. Sesame HA, Castro JA, and Gillette JR: Studies on the destruc18. Snedecor GW, Cochran WG: Statistical Methods. Ames, Iowa, tion of liver microsomal cytochrome P-450 by carbon tetrachloThe Iowa State University Press, 1967 ride administration. Biochem Pharmacol 17:1759-1768, 1968 19. Dixon MF, Dixon B, Aparicio SR, et al: Experimental paraceta- 34. Willson RA, Hart FE: Experimental hepatic injury: the sequenmol-induced hepatic necrosis: a light and electronmicroscope, tial changes in drug metabolizing enzyme activities after adminand histochemical study. J Pathol116:17-29, 1975 istration of acetaminophen. Res Commun Chern Pathol Pharma20. Prescott LF, Wright N, Roscoe P, et al: Plasma-paracetamol col (in press) half-life and hepatic necrosis in patients with paracetamol over- 35. Klotz U, Avant GR, Hoyumpa A, et al: The effects of age and dosage. Lancet 1:519-522, 1971 liver disease on the disposition and elimination of diazepam in 21. Reynolds ES, Moslen MT, Szabo S, et al: Vinyl chloride-induced adult man. J Clin Invest 55:347-359, 1975 deactivation of cytochrome P-450 and other components of the 36. Breimer DD, Zilly W, Richter E: Pharmacokinetics of hexobarbiliver mixed function oxidase system: an in vivo study. Res Comtal in acute hepatitis and after apparent recovery. Clin Pharmamun Chern Pathol Pharmacol 12:685-694, 1975 cal Ther 18:433-440, 1975 22. Wanson JC, Drochmans P, May C, et al: Isolation ofcentrolobu- 37. Mawer CE, Miller NE, Turnburg LA: Metabolism of amylobarlar and perilobular hepatocytes after phenobarbital treatment. J bitone in patients with chronic liver disease. Br J Pharmacol Cell Bioi 66:23-41, 1975 44:549-560, 1972 23. Hutterer F, Greim H, Trulzsch D, et al: Microsomal biotransfor- 38. Branch RA, Herbert CM, Read AE: Determinations of serum mation system in cholestasis. In Progress in Liver Diseases, vol antipyrine half-lives in patients with liver disease. Gut 14:5694. Edited by H Popper, F Schaffner. New York, Grune & Strat573, 1973 ton, 1972, p 151-171 39. Andreasen PB, Ranek K, Statlant BE, et al: Clearance of anti24. Shull HJ, Wilkinson GR, Johnson R, et al: Normal disposition of pyrine-dependence of quantitative liver function. Eur J Clin oxazepam in acute hepatitis and cirrhosis. Ann Intern Med Invest 4:129- 134, 1974 84:420-425, 1976 40. Schenker S, Hoyumpa AM, Wilkinson GR: The effect of paren25. Smuckler E, Arrhenius E, Hultin T: Alterations in microsomal chymalliver disease on the disposition and elimination of sedaelectron transport, oxidative N-demethylation and azo-dye tives and analgesics. Med Clin North Am 59:887-896, 1975 cleavage in carbon tetrachloride and dimethylnitrosamine-in- 41. Davidson AR, Rojas-Bueno A, Thompson RPH, et al: Unconjuduced liver injury. Biochem J 103:55-64, 1967 gated hyperbilirubinemia in paracetamolliver damage: clinical 26. Yu CA, Gunsalus IC: Cytochrome P-450. II. Interconversion with and experimental studies (abstr) Digest ion 8:444, 1973 P-420. J Bioi Chern 249:102-106, 1974 42. Davis M, Ideno G, Ha-rison NG, et al: Early inhibition ofhepatic 27. Dixon MF, Fulker MJ, Walker BE, et al: Serum transaminase bilirubin conjugation after paracetamol (acetaminophen) adminlevels after experimental paracetamol-induced hepatic necrosis. istration in the rat. Digestion 13:42-48, 1975 43. Martin JR, Vierling JM, Wolkoff AW, et al: Abnormal hepatic Gut 16:800-807, 1975 transport of indocyanine green in Gilbert's syndrome. Gastroen28. Hoensch H, Woo CH, Raffin SB, et al: Oxidative metabolism of foreign compounds in rat small intestine: cellular localization terology 70:385-391, 1976 and dependence on dietary iron. Gastroengerology 70:1063-1070, · 44. Maines MD, Anders MW, Muller-Eberh ard U : Studies on heme transfer from microsomal hemo-protein to heme-binding plasma 1976 29. Pantuck EJ, Hsiad KC, Long WD, et al: Stimulating effect of proteins. Mol Pharmacol 10:204-213, 1974 vegetables on intestinal drug metabolism in the rat. J Pharma- 45. Lauterburg BH, Bircher J: Expiratory measurement of maximal cal Exp Ther 198:278-283, 1976 aminopyrine demethylase in vivo: effect of phenobarbital, par30. Mitchell JR, Jollow DJ: Metabolic activation of drugs to toxic tial hepatectomy, portacaval shunt and bile duct ligation in the rat. J Pharmacol Exp Ther 196:501-509, 1976 substances. Gastroenterology 68:392-410, 1975 31. Jollow DJ, Thorgeirsson SS, Potter WZ, et al: Acetaminophen- 46. Bircher J, Kupfer A, Gikalov I, et al: Aminopyrine demethylation measured by breath a nalysis in cirrhosis. Clin Pharmacol induced hepatic necrosis. VI. Metabolic disposition of toxic and Ther 20:484-492, 1976 non-toxic doses of acetaminophen. Pharmacology 12:251-271, 47. Hepner GW, Vesell ES: Assessment of aminopyrine metabolism 1974 32. Willson RA, Hart FE: The effect of parenteral dimethyl sulfoxide in man by breath analysis after oral administration of 14C(DMSO) on drug metabolizing enzyme activities. Biochem Pharaminopyrine. Effects of phenobarbital, disulfiram and portal cirrhosis. N Engl J Med 291:1384-1388, 1974 macol16:59-71, 1977
GASTROENTEROLOGY 73:691-696, 1977 Copyright © 1977 by the American Gastroenterological Association
Printed in U.SA .
EFFECT OF EXPERIMENTAL HEPATIC INJURY ON IN VITRO DRUGMETABOLIZING ENZYME ACTIVITIES IN THE RAT R. A.
WILLSON,
M.D.,
AND
F. E.
HART,
B.S.
Division of Gastroenterology, University of Washington , Seattle, Washington
Experimental hepatic necrosis was induced in phenobarbital pretreated rats by means of the intraperitoneal administration of an acetaminophen-dimethyl sulfoxide mixture over a dosage range of 0.3 to 1.5 g per kg. At lower doses, cytochrome P-450 concentration and the specific activities of aminopyrine demethylase and bilirubin glucuronyl transferase increased, whereas aniline hydroxylase activity was mildly decreased. In contrast, &t higher doses, cytochrome P-450 concentration and the specific activities of all of the enzymes were decreased significantly (P < 0.01), as compared with control rats. The diminished activities, however, were generally modest, not uniform for all enzymes assayed, and correlated poorly with histological necrosis and routine laboratory tests. When necrosis was severe, a prominent peak was detected at 420 nm, and this was associated with a more consequential decrease in all of the enzyme activities. When the total in vitro hepatic drug-metabolizing enzyme capacity was calculated, only the cytochrome P-450 content and aminopyrine demethylase activity were significantly decreased at the 1.5-g per kg dose level. It is concluded that in acute liver disease changes in the hepatic in vitro drug-metabolizing enzyme capacity may not be closely related to cellular necrosis, per se, and the degree of change in enzyme activities will vary from one enzyme system to another. These findings may explain, in part, the often inconsistent alterations in the disposition and elimination of drugs described in associated liver diseases. The disposition of drugs, whose major biotransformation is within the. liver, may be significantly altered with associated hepatic disease; however, some drugs may not be affected, and the degree of alteration may be variable. 2 Two recognized factors that may in part account for these inconsistencies are the complex metabolic pathways involved in drug biotransformation, and the heterogeneity of the patients studied as regards stage or severity of liver disease. In acute hepatic injury, a reduced drug clearance may be related to a number of pharmacokinetic factors, however, an impaired extraction by the liver may be a principal cause. 3 Both diminished uptake and/or rate of metabolism would result in a reduced drug extraction ratio. Concerning the rate of metabolism, some in vitro drugmetabolizing enzyme activities have been assayed on human liver biopsy specimens, however, their activities
were not greatly reduced unless the extent of necrosis was severe,4 and not all enzyme activities were uniformly affected. Further, a poor correlation between in vitro drug-metabolizing enzyme activities and routine "liver function" tests has been reported in acute hepatitis, both in the initial episode and during resolution. 5 Because of these limited observations, it was thought that a systematic evaluation of some selected in vitro drug-metabolizing enzyme activities under uniform experimental conditions might help to clarify the relative role of the in vitro drug-metabolizing enzyme capacity in altered drug disposition when associated with acute liver injury. Consequently, the purpose of the present study was to investigate the relationship between in vitro drug-metabolizing enzyme activities, liver necrosis, and routine laboratory tests in the rat with acetaminophen-induced hepatic injury.
Received July 6, 1976. Accepted April 14, 1977. Materials and Methods This paper was presented in part at the Annual Meeting of the Male Sprague-Dawley rats (240 to 260 g) were used in all American Association for the Study of Liver Diseases, Chicago, Illinois, November 4 and 5, 1975, and has been published in abstract experiments. They had free access to food (Purina rat chow, Ralston Purina Company; St. Louis, Mo.) and water, and were form.' Address requests for reprints to: Richard A. Willson, M.D., Har- caged in a constant temperature room (22°C) with alternating borview Medica l Center, 325 9th Avenue, Seattle, Washing- 12 hr of light and darkness. All studies were done in the early morning. ton 98104. This study was supported in part by Grant AM 17813 from the Preparation of the hepatotoxin. Acetaminophen (4'hydroxyUnited States Public Health Service. acetanilide, Eastman Organic Chemicals, Rochester, N.Y.), a Dr. Willson is a recipient of Academic Career Development known liver toxin, ~ was assessed for purity by thin layer Award AM-00151 from the National Institute of Arthritis, Metabo- chromatography on Silica Gel G in ethyl acetate-methanollism and Digestive Diseases. water-acetic acid, 60:30:9:1, v/v. 7 In our experience, the dos691
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WILLSON AND HART
ages of acetaminophen that we planned to utilize were not consistently soluble in warm saline at alkaline pH. 8 Consequently, 0.5 ml of dimethyl sulfoxide (DMSO) (Fisher Scientific Company, Fairlawn, N.J., spectral grade) was used as a solvent in the preparation of the acetaminophen for intraperitoneal administration. The DMSO was chromatographed in the same system and had an R distinct from that of acetaminophen. The acetaminophen-DMSO mixture was assessed chromatographically also and produced R values corresponding to their respective components with no evidence for a drugDMSO complex, as has been reported for some compounds. 9 To investigate further the possibility of drug interaction with DMSO, gas liquid chromatography-mass spectroscopy (kindly performed by Dr. V. Raisys, Associate Director, Toxicology Laboratory, Harborview Medical Center, Seattle, Washington) was performed on the mixtures. The sensitivity of this analysis was such that it would detect the mixture down to 100-ng quantities. Again, each compound was pure and there was no evidence for drug-DMSO interaction. Animal preparation. Because microsomal enzyme induction enhances the h epatic necrosis caused by acetaminophen, 8 all rats were pretreated with phenobarbital (75 mg per kg per day) intraperitoneally for 4 days before the administration of the acetaminophen. Then the rats were divided randomly into two groups. The first was given 0.5 ml of DMSO intraperitoneally and are considered as the treatment controls. The second received the acetaminophen mixture intraperitoneally over a dosage range of 0.3 to 1.5 g per kg and are considered as the treatment group. Preparation of tissue fractions. The rats were fasted overnight and killed by decapitation approximately 72 hr after the acetaminophen administration. Mter decapitation, the liver was perfused in situ with 75 ml of ice-cold 0.15 M sodium chloride, and then excised, weighed, and biopsied. One gram of t he liver was then homogenized with 9 ml of0.25 M sucrose in 0.001 M ethylenediaminetetraacetate (EDTA),pH 7.4, with a glass pestle and homogenizing tube. Six grams of the remaining liver were homogenized with 12 ml of 0.01 M sodiumpotassium phosphate with 1.15% potassium chloride, pH 7.4. Microsomes from both homogenates were prepared by differential centrifugation at 105,000 x g for 1 hr in a Beckman L265 B ultracentrifuge (Beckman Instruments, Inc. , Fullerton, Calif.). The microsomal pellets resulting from the sucrose homogenates were resuspended in 5 ml of 0.25 M sucroseEDTA and used for the glucuronyl transferase assay. The pellets resulting from the sodium-potassium phosphate homogenates were resuspended in 10 ml of 0.05 M sodium-potassium phosphate and were used for the aminopyrine demethylase, aniline hydroxylase, and cytochrome P-450 assays. Enzyme assay. Protein concentrations of all microsomal suspensions were determined by the method of Lowry et al. 10 using bovine serum albumin as standard. Concentrations of protein were adjusted for optimal conditions. Repeat Lowry determinations were done on the adjusted samples. Incubations were carried out in a 37oC shaking water bath for all assays. Aminopyrine demethylase activity was assayed according to the method given by Mazel 11 with the following modifications in order to give maximal activity: 20 p.moles of aminopyrine, 2.5 units of glucose-6-phosphate dehydrogenase, and 5 mg of microsomal protein were added to the incubation. The formaldehyde produced was estimated by the method of Nash. 12 Aniline hydroxylase activity was assayed according to the method given by Mazel 13 with the following modifications in order to give maximal activity: 20 p.moles of aniline-HCl, 1 p.mole ofNADP, 2.5 units ofglucose-6-phosphate dehydrogenase, and 10 mg of microsomal protein were added to the incubation mixture. Bilirubin glucuronyl transferase activity
Vol. 73, No.4, Part 1
was assayed utilizing the method of Black et al. 14 Cytochrome P-450 content was measured by the method of Omura and Sato 15 using a Beckman 25 spectrophotometer in the sp lit beam mode. Liver histology . Initially, liver specimens were taken from four arbitrary areas in the liver and evaluated for their uniformity in the representation of necrosis. Because there was little difference among the four areas sampled, only one section was taken for the remainder of the study. The specimens were fixed in formalin and stained with hematoxylin and eosin. The sections were graded by light microscopy into minor necrosis (grade I), moderate necrosis (grade II), and severe necrosis (grade III).'" Biochemical indicators of liver injury. Serum bilirubin concentrations were assessed spectrophotometrically as described by Malloy and Evelyn. 17 Serum glutamic oxaletic transaminase (SGOT) activities were assayed by the colorimetric method of Sigma at 505 nm (Sigma Chemical Company, St. Louis, Mo.). Blood samples were drawn for these analyses before treatment and at the time of death. Expression of data. Microsomal drug-metabolizing enzyme specific activities are expressed as product formed per unit time per milligram of microsomal protein, and as an estimate of a hepatic drug-metabolizing enzyme capacity by the product formed per unit time per total liver weight per 100 g of body weight. Analysis of data . Statistical indices were calculated by standard methods and are expressed as the mean ± SEM. A ttest of the difference between two sample means was utilized to assess significance between parameters. 18
Results and Discussion Effect of acetaminophen dosage upon mortality and hepatic necrosis. Table 1 illustrates the relationship between acetaminophen dosage, hepatic necrosis, and mortality. At the 300-mg per kg dose schedule·there was neither hepatic necrosis nor rat death. Both mortality and the percentage of rats with histological necrosis showed a commensurate increase with dose up to the 1200-mg per kg level. There was, however, considerable variation in the presence and extent of necrosis between rats, and this has been observed by others. 19 At the 1500-
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October 1977
mg per kg dose, the percentage of animals demonstrating histological necrosis decreased, although the mortality continued to increase. This suggested that at the highest dose, death was related to causes other than hepatic necrosis. Such a conclusion was further supported by the fall in serum transaminase at this dose schedule (fig. 2). Complete autopsies were not done on the rats so other organ damage is only postulated. A previous report also noted a similar discordance between mortality and hepatic necrosis, and the cause of death was not apparent. 8 Acetaminophen-induced hypoglycemia has been postulated as a cause, 20 but we did not assess blood glucose levels. Death of the rats usually occurred within 12 to 24 hr after the acetaminophen administration. Effect of acetaminophen dosage upon drug-metabolizing enzyme specific activities and hepatic drug metabolizing enzyme capacity. All acetaminophen-treated rats lost weight as the dosage schedule increased, and at the higher doses the liver weight increased. The calculated gram liver weight per 100 gram of body weight (mean ± SEM) was as follows: control (3.51 ± 0.25), 300 mg per kg (3.45 ± 0.17), 600 mg per kg (3.92 ± 0.35) 900 mg per kg (3.68 ± 0.35), 1200 mg per kg (4.24 ± 0.50), 1500 mg per kg (4.28 ± 0.58). Table 2 shows the change in microsomal protein concentrations and the enzyme-specific activities over the corresponding dose schedule. The microsomal protein concentration represents our yield following ultra centrifugation. There was a trend for the enzyme activities to increase over the lower doses (300 and 600 mg per kg), however, these changes were not significant. As the acetaminophen dose schedule increased, the cytochrome P-450 concentration and the aminopyrine demethylase activity decreased, and at the 1500-mg per kg dose, both were approximately 60% of control value. In contrast, the aniline hydroxylase and TABLE 1. Effect of acetaminophen dosage upon hepatic necrosis and mortality in rats Rats with histologiMortality Acetaminophen dose cal necrosis mg/kg % % 300 (6)" 0 0 600 (14) 21 0 900 (22) 30 9 1200 (29) 40 31 1500 (27) 31 52 a
Number of animals in parentheses.
Treatment mg!kg
Control 300 600 900 1200 1500
glucuronyl transferase activities were not as affected at the 1500-mg per kg dose level, both being about 80% of control value. Therefore, all enzyme specific activities were not decreased equally, and the diminished activities, although significant, were generally modest. The estimation of the hepatic drug-metabolizing enzyme capacity (table 3) revealed a similar trend for the enzyme activities to increase at the lower dosage levels, and at the 1500-mg per kg dose, only the cytochrome P-450 and aminopyrine demethylase were significantly decreased. The nonuniformity in the decrease of enzyme activities has previously been reported. Certain hydroxylase enzymes are less affected by experimental necrosis, 21 and this may be related to the regionalization of drug-metabolizing enzyme activities within the hepatic lobule. 22 Further, experimental cholestasis studies have shown that the decrease in aniline hydroxylase activity was less than the aminopyrine demethylase activity, and this was related to the involvement of different binding sites on the cytochrome P-450 molecule. 23 Lastly, it has recently been reported that the glucuronidation pathway in drug disposition may not be as affected by liver injury as the biotransformation pathways via the mixed function oxygenase system. 24 At the 900-, 1200-, and 1500-mg dose levels, there was a prominent spectral peak at 420 nm (fig. 1) noted in 16,
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300 500 900 1200 1500 Acetaminophen dosage, mg I kg
FIG. 2. The changes in serum transaminase (SGOT) over the acetaminophen dose schedule. Ll.SGOT represents the mean difference between pretreatment and post-treatment (72 hr) values for SGOT in the same animals. The number of animals at each dose is the same as that given in table 2.
TABLE 2. Effect of acetaminophen dose on drug-metabolizing Aminopyrine demethylMicrosomal protein Cytochrome P-450 ase HCHO formed!mg nmoles mg/g liver wt nmoles!mg protein protein/min 36.82 ± 1. 73 (33) 1.70 ± 0.07 (28) 5.45 ± 0.43 (31) 43.10 ± 3.33 (4) 1.93 ± 0.11 (4) 6.29 ± 0.16 (4) 45.93 ± 2.54b (14) 1.81 ± 0.09 (8) 5.97 ± 0.50 (14) 34.76 ± 1.28 (19) 1.26 ± 0.1~ (15) 4.55 ± 0.56 (15) 37.15 ± 1.37 (19) 1.21 ± 0.08b (19) 4.09 ± 0.35C (18) 33.16 ± 1.41 (13) 1.05 ± 0.07b (13) 3.36 ± 0.34b (13)
Values given are the mean± SEM with number of observations in parentheses. P < 0.01 as compared with control. c p < 0.05 as compared with control. a
b
693
DRUG-METABOLIZING ENZYMES IN HEPATIC INJURY
enzyme activities•
Aniline hydroxylase
Glucuronyl transferase
nmoles p-aminophenol formed/mg protein/min 1.05 ± 0.02 (27) 1.00 ± 0.02 (4) 0.89 ± 0.03 (4) 0.90 ± 0.06b (19) 0.97 ± 0.06 (19) 0.87 ± 0.06b (13)
pg bilirubin formed!mg protein/min 1.29 ± 0.04 (28) 1.51 ± 0.09 (4) 1.51 ± 0.09 (10) 0.95 ± 0.08b (14) 1.10 ± o.w (18) 1.02 ± 0.07b (13)
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WILLSON AND HART
16, and 25% of the rats, respectively. This spectral peak was associated with grade II and grade III hepatic necrosis, and · a more consequential decrease of the hepatic drug-metabolizing enzyme capacity as compared with their mean levels for these three dose schedules (table 4). For example, at the 900-mg per kg dose level, the cytochrome P-450 content and the activities of aminopyrine demethylase, aniline hydroxylase and glucuronyl transferase were 36, 47, 46, and 46% of their control values, respectively. Therefore, with severe necrosis all enzyme activities were similarly decreased. The alteration of cytochrome P-450 to a probable P-420 degradation product has been reported previously. 25• 26 The relationship between drug-metabolizing enzyme activities and hepatic necrosis. Although the aminopyrine demethylase activity and cytochrome P-450 concentration diminished with increasing doses of acetaminophen, their decrease did not correlate well with the estimated extent of necrosis. At the 900-, 1200-, and 1500-mg dose levels, the correlation between cytochrome P-450 and hepatic necrosis was -0.77, -0.56, and -0.55, respectively; similar values were found with the aminopyrine demethylase activity. This rather poor correlation with cellular necrosis could, in part, reflect the probable inaccuracy of the qualitative assessment of the tissue injury. Utilizing several techniques for assessing this, Dixon found considerable variation in the extent of necrosis from different parts of the rat liver, even with multiple tissue blocksY We observed that the extent of hepatic necrosis when present was generally modest and this is in agreement with a previous report over a similar dose range. 8 The route of administration may be important as regards development of hepatic necrosis, in that oral acetaminophen19• 27 appears to induce hepatic necrosis more readily than does the intraperitoneal route, 8 however, the dose administered orally TABLE
Treatment
3. Effect of acetaminophen dose on drug-metabolizing enzyme capacity of the liver'
Cytochrome P-450 nmoles/totalliver wt/100 g body wt
mg/kg
Control 300 600 900 1200 1500
216.23 282.67 328.58 150.92 190.23 148.27
is generally larger. An additional explanation could be that the biotransformation of drugs within the enterocyte of the proximal small intestine28• 29 may play a role in drug-induced liver injury, similar to the now recognized role of hepatic drug biotransformation. 30 The rat is recognized to be more resistant to acetaminophen than other species3 1 and to have a variable hepatic injury response. 8 In addition, we have reported that DMSO diminishes the activity of the mixed function oxygenase enzyme system in the phenobarbital pretreated rat,32 and this reduced activity would lessen the toxicity of acetaminophen. Alternatively, previous studies suggest that perhaps a poor relationship with necrosis should not be unexpected, as some drug-metabolizing enzyme activities are diminished in cholestasis, 23 where hepatocellular necrosis is not prominent; some in vitro enzyme activities are decreased after CC1 4 administration without cellular necrosis; 3 and some in vitro enzyme activities are not decreased even when hepatic necrosis is quite marked.4 Finally, in a 3-day sequential study, we found that all enzyme specific activities were diminished by 24 hr without evidence for histological necrosis. 34 The relationship between drug-metabolizing enzyme activities and routine laboratory tests . Figure 2 illustrates the changes in serum oxalacetic transaminase levels over the acetaminophen dose range, with the greatest elevation being at the 1200-mg per kg dose, followed by a decrease at the 1500-mg dose level. Therefore, at the highest dose, the SGOT elevation was minid mal, whereas both the cytochrome P-450 concentration and the aminopyrine demethylase activity were at their lowest levels. It is apparent then that the relationship was limited. The changes in serum transaminase, however, did reflect hepatic necrosisn as the correlation coefficient between the changes in transaminase and
Aniline hydroxylase Aminopyrine demethylase nmoles HCHO formed/min/total liver nmoles p-aminophenol formed/ wt/100 g body wt min/total liver wt/100 g body wt
± 85.80 (28) ± 23.18 (4) ± 92.40 (8)
728.87 889.58 1041.14 589.05 643.33 447.22
± 56.54b (15) ± 63.30 (19) ± 40.45C (13)
68.18 (31) 151.59 (4) 109.57" (14) 69.30 (15) 64.32 (18) ± 35.13" (13) ± ± ± ± ±
131.43 154.19 167.45 114.10 152.16 119.95
Glucurony I transferase pg bilirubin formed/min/total liver wt/100 g body wt
111.27 134.48 178.80 86.25 126.18 102.28
7.28 (27) 14.48 (4) 14.41 (4) 8.07 (19) 9.94 (19) ± 8.92 (13) ± ± ± ± ±
± 7.91 (28) ± 15.94 (4)
± 16.75 (10) ± 9.74 (14) ± 6.31 (18) ± 9.60 (13)
• Values given are the mean ± SEM with number of observations in parentheses. P < 0.05 as compared with control. c P < 0.01 as compared with control. b
TABLE
4. Drug-metabolizing enzyme capacities in the animals in which a prominent spectral p eak at 420 nm was detected•
Treatment
Bilirubin
SGOT"
mg/100 ml
Control 900 1200 1500
0.1 0.27 0.17 0.20
8 497 1831 72
Aminopyrine deAniline hydroxylase Glucuroriyl transmethylase ferase . nmoles HCHO formed/ nmoles p-aminophenol pg bilirubin formed/ nm~l~~/to~lJwe~ wtl min/total liver wt/100 formed/min/total liver min/total liver wt/100 g 0 Yw g body wt wt/100 g body wt g body wt Cytochrome P-450
216.23 76.26 118.11 98.94
728.87 346.03 341.05 264.76
131.43 60.65 80.20 71.22
111.27 51.19 90.14 50.38
• The drug-metabolizing enzyme activities are mean values tak en from table 3. Values given represent the mean with number of observations in parentheses. b 6.SGOT represents mean difference between pretreatment and post-treatment (72 hr) values for SGOT in the same animals.
DRUG-METABOLIZING ENZYMES IN HEPATIC INJURY
October 1977
histological necrosis 'Vas r = 0. 74, a value similar to that reported by Dixon et al. 27 The poor relationship between routine laboratory tests and the altered disposition of drugs has been reported by a number of authors35· 36 and only with severe liver disease has a correlation been noted with serum albumin and prothrombin time. 37..:39 The dissociation of these tests and clearance of drugs after clinical recovery from acute hepatic disease has further suggested that routine laboratory tests may bear little association with the liver's capacity to metabolize drugs.40 The relationship of serum bilirubin levels, primarily unconjugated bilirubin, with cytochrome P-450 concentration and glucuronyl transferase activity is illustrated in figure 3. The elevation of serum bilirubin was small, but it did rise with the commensurate fall in glucuronyl transferase activity. It has been proposed that the unconjugated hyperbilirubinemia seen early in clinical acetaminophen overdose may be attributable to a decreased glucuronyl transferase activityY In addition, experimental studies in rats suggest that hepatic glucuronide conjugation is impaired early after acetaminophen overdose. 42 Other factors may be involved, however, as it is recognized that the in vitro assay of glucuronal transferase activity does not always correlate well with serum bilirubin levels. 43 As shown in figure3, the cytochrome P-450 concentration also decreased and the serum bilirubin was elevated in the group of animals that demonstrated the 420 nm spectral peak (table 4). It is possible that this 420 peak represents a degraded cytochrome P-450, and it has been suggested that the degraded hemeprotein would be catabolized via hemoxygenase to unconjugated bilirubin. 44 Consequently, our findings indicate that the unconjugated hyperbilirubinemia seen with severe hepatocellular necrosis could be related to both the increased degradation of cytochrome P-450 and the diminished glucuronyl transferase activity. Application to human disease . The extrapolation of in vitro drug-metabolizing enzyme activities to the whole liver's capacity in vivo is difficult, as the in vitro assays are done under quite artificial circumstances and they, 120
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695
of course, do not reflect alterations in the various pharmacokinetic factors that influence drug disposition in vivo. The results of this study, however, do suggest that in acute liver disease, changes in the hepatic in vitro drug-metabolizing enzyme capacity may not be closely related to cellular necrosis, per se, and the degree of change iri. enzyme a<;:tiyities will vary from one enzyme system to another. These findings may explain, in part, the often inconsistent alterations in disposition and elimination of drugs in associated liver disease. The recent reports utilizing a noninvasive breath analysis method for the assessment of drug disposition in liver disease 45 -47 may provide more direct information on altered in vivo drug metabolism and further should provide useful correlations with in vitro drug-metabolizing enzyme activities. REFERENCES 1. Willson RA, Hart FE: Effect of experimental hepatic injury on microsomal drug metabolizing activity (abstr). Gastroenterology 69:880, 1975 2. Wilkinson GR, Schenker S: Drug disposition and liver disease. Drug Metab Rev 4:139-175, 1975 3. McHorse TS, Wilkinson GR, Johnson RF, et al: Effect of acute viral hepatitis in man on the disposition and elimination of meperidine. Gastroenterology 68:775-780, 1975 4. Schoene B, Fleischmann RA, Remmer H, et al: Determination of drug metabolizing enzymes in needle biopsies of human liver. Eur J Clin Pharmacal 4:65-73, 1972 5. Doshi J, Luisada-Opper A, Leevy CM: Microsomal pentobarbital hydroxylase activity in acute viral hepatitis. Proc Soc Exp Biol Med 140:492-495, 1972 6. Dixon MF, Nimmo J, Prescott LF: Experimental paracetamolinduced hepatic necrosis: a histopathological study. J Pathol 103:225-229, 1971 7. Cummings AJ, King ML, Martin BK: A kinetic study of drug elimination: the excretion of paracetamol and its metabolites in man. Br J Pharmacal Chemother 29:150-157, 1967 8. Mitchell JR, Jollow DJ, Potter WZ, et al: Acetaminophen-induced hepatic necrosis. I. Role of drug metabolism. J Pharmacal Exp Ther 187:185-194, 1973 9. Levine WG: Effect of dimethyl sulfoxide on the hepatic disposition of chemical carcinogens. Ann NY Acad Sci 243:185-193, 1975 10. Lowry OH, Rosebrough NJ, Farr AL, et al: Protein measurement with the Falin phenol reagent. J Biol Chern 193:265-275, 1951 11. Mazel P: Experiments illustrating drug metabolism in vitro. In Fundamentals of Drug Metabolism and Disposition. Edited by BN LaDu, HG Mandel, EL Way. Baltimore, Williams & Wilkins Co, 1971, p 546-550 12. Nash T: The colorimetric estimation of formaldehyde by means of the Hantzsch reaction. Biochem J 55:416-421, 1953 13. Mazel P: Experiments illustrating drug metabolism in vitro. In Fundamentals of Drug Metabolism and Disposition. Edited by BN LaDu, HG Mandel , EL Way. Baltimore, Williams & Wilkins Co, 1971, p 569-572 14. Black M, Billing B, Heirwegh KPM: Determination of bilirubin UDP-glucuronyl transferase activity in needle-biopsy specimens of human liver. Clin Chim Acta 29:27-35, 1970 15. Omura T, Sato R: The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J Biol Chern 239:2370-2378, 1964 16. Chalkley HW: Method for the quantitative morphologic analysis of tissues. J Natl Cancer lnst 4:47-53, 1943 17. Malloy HT, Evelyn K: The determination of bilirubin with the
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