Lysyl Oxidase and Collagenase in Experimental Acute and Chronic Liver Injury

Transcription

1 GASTROENTEROLOGY 1982;82: Lysyl Oxidase and Collagenase in Experimental Acute and Chronic Liver Injury EDWARD A. CARTER, MARY JANE McCARRON, ELLIOT ALPERT, and KURT J. ISSELBACHER The Department of Medicine, Harvard Medical School; Gastrointestinal Unit of the Massachusetts General Hospital, Boston, Massachusetts Lysyl oxidase and collagenase activities were measured in experimental acute and chronic liver injury in mice and rats, and correlated with collagen synthesis and accumulation. Acute liver injury was induced in mice and rats by a single dose of carbon tetrachloride given by gavage, and also in mice by a single injection of murine hepatitis virus. Chronic liver injury was induced in rats by repeated injectiqns-o[carbon tetrachloride. Elevated plasma glutamic oxaloacetic transaminase levels, increased hepatic prolyl hydroxylase activity, and increased synthesis of collagen-bound hepatic hydroxyproline occurred in animals with acute as well as with chronic liver injury. However, only chronic liver injury appeared to be associated with fibrosis, increased collagen-bound hydroxyproline content, increased hepatic lysyl oxidase and collagenase activities, as well as with increased serum lysyl oxidase activity. These data suggest that lysyl oxidase and collagenase may play an important role in the collagen accumulation associated with hepatic fibrosis. In normal liver, collagen is found only in small amounts (approximately 4%) (1), whereas in hepatic cirrhosis there is an increase in connective tissue (defined as hepatic fibrosis) that contains newly Received January 13, Accepted October 23, Address requests for reprints to: Dr. Edward A. Carter, Gastrointestinal Unit, Massachusetts General Hospital, Boston, Massachusetts This study was supported by grants from the National Institutes of Health (AA00239, AM01392, AM03014) and the National Institute of General Medical Science (GM07753). The authors are indebted to Drs. Stephen Krane and Jean Michael Dayer, Massachusetts General Hospital, and Dr. Herbert Kagan, Boston University, School of Medicine, for invaluable discussions and advice. They also thank Mr. Michael Byrne for performing the amino acid analyses by the American Gastroenterological Association /82/ $02.50 formed collagen fibers (1-5). Although the fibrotic liver contains more collagen than the normal liver (6-13), the mechanism or mechanisms involved in this accumulation are not clear. In order to attempt to elucidate the mechanism or mechanisms of the fibrosis, one needs to examine the steps in the biosynthesis of collagen (14,15). Amino acids are first activated and then incorporated into procollagen in the ribosomes. The procollagen is acted on by a number of intracellular enzymes, including prolyl hydroxylase, lysyl hydroxylase, and glycosyltransferase. The procollagen is then secreted into the extracellular space where the "pro" portions of each end are cleaved off by pro collagen peptidases. The remaining collagen spontaneously forms aggregated collagen fibrils, which are then formed into stable cross-linked collagen by the enzymatic action of lysyl oxidase. The aggregated collagen fibrils as well as the cross-linked collagen (and possibly procollagen) can subsequently be degraded by collagenase. Recent studies have suggested that alterations of either hepatic lysyl oxidase (16) or hepatic collagenase (17-26) activities may be involved in the collagen accumulation associated with fibrosis in chronic liver injury. The purpose of the present investigation was to measure both lysyl oxidase and collagenase activities in acute as well as chronic liver injury, and to correlate these enzyme activities with overall hepatic collagen accumulation. Methods Animals Acute liver Injury was induced in mice (CD-l strain, Charles River Breeding Laboratories, Wilmington, Mass.) by two methods. In the first method, male mice weighing 20 g were given a single dose of carbon tetrachloride by gavage (3 mllkg, 40% vol/vol in mineral oil).

2 March 1982 LYSYL OXIDASE AND COLLAGENASE IN LIVER INJURY 527 Control mice received an equal volume of mineral oil. In the second method. acute liver injury was induced by injecting 21 day-old female mice intraperitoneally with 16 complement fixation units of murine hepatitis virus (MHV-A-59. M.A. Bioproducts. Wakersville. Md.). Control mice received an equal volume of the media (NCTC-109. M.A. Bioproducts) that contained no virus or cellular material. Acute and chronic liver injury was induced in female Sprague-Dawley rats weighing between 100 and 200 g (Charles River Breeding Laboratories). Acute liver injury was achieved by admiriistration of a single dose of carbon tetrachloride by gavage (3 mllkg. 40% vol/vol in mineral oil); control rats received an equal volume of mineral oil. In other experiments partial (66%) hepatectomy was first performed by standard techniques under ether anesthesia (27). Chronic liver injury was induced in rats by repeated i.p. injections of carbon tetrachloride (0.75 mllkg. 15% voll vol in mineral oil) three times a week (on Monday. Wednesday. and Friday) for a total of 15 injections; control rats were injected with an equal volume of mineral oil. Both treated and control rats were killed on the Monday after the last injection on Friday. Histology Liver samples of mice or rats were fixed in formalin. and prepared for histologic examination by routine methods. The fixed sections of liver samples were stained with Mallory trichrome. Plasma Glutamic Oxaloacetic Transaminase Plasma glutamic oxaloacetic transaminase (GOT; aspartate aminotransferase) activities were determined as described previously (28). Three milliliters of a reconstituted GOT diagnostic kit (Sigma Chemicai Co.. #55-UV. St. Louis. Mo.) was preincubated in a glass cuvette in a Gilford spectrophotometer (Gilford Instrumerit Laboratories. Inc. Oberlin. Oh.) for 10 min at 37 C. The reaction in the cuvette was then initiated by the addition of 0.01 ml of plasma. The activity was expressed in International units per 0.01 ml of plasma. based on calculations using Sigma Enzyme controls. 2-E and 2-N (Sigma Chemical Co.). Prolyl Hydroxylase Prolyl hydroxylase activity was measured in mice or rat liver homogenates according to the procedure used previously in this laboratory to measure prolyl hydroxy" lase activity in human liver biopsy specimens. (45). One gram of liver tissue was homogenized in 50 ml of 100 mm Tris-HCl buffer. ph 7.4. by using a Potter-Elvehjem homogenizer (5 strokes. full speed). One-half milliliter of tissue homogenate was incubated in a 25-ml Erlenmeyer flask for 1 h with shaking (100 strokes/min) under air with 1.5 ml of buffer (1 mm ferrous ammonium sulfate. 1 mm a ketoglutarate. 5 mm ascorbic acid. 133 mm Tris-HC!, ph 7.4) and 0.5 ml 3H-cbllagen substrate containing about cpm prepared from 17-day-old chick calvaria (29). The chick calvaria had been previously labeled with [3,4-3H]proline (25-50 Ci/mmol, New England Nuclear Co. Boston. Mass.]. The reaction was terminated by the addition of 0.2 ml 50% trichloroacetic acid (TCA). The assay mixture was then centrifuged,at 9000 g for 10 min. The blank for the reactions was a set of tubes to which the 50% TCA was added at zero time. The counts that were found in these tubes were subtracted from tubes incubated 1 h at 37 C. The supernatant was decanted and the 3H20 (formed by the action of the prolyl hydroxylase) was vacuum distilled into counting vials (29). After the contents of the counting vials had thawed. 10 ml of Aqtiasol 2 (New England Nuclear Co.) was added to each of the counting vials. and the radioactivity of the contents was measured by liquid scintillation spectrometry. The recovery of the 3H20 was determined in one set of experiments. Threetenths of a milliliter of 3H20 containing 1000 cpm was added to the assay mixture in the absence of the collagen substrate. and the distillation procedure carried out. Under these conditions 80%-90% of the 3H20 counts were recovered.. Triton X-lOO has been used to measure the activity of prolyl hydroxylase activity in membranous tissue fractions (46). We did not use this detergent because preliminary experiments in this laboratory suggested that the addition of Triton X-100 to our tissue homogenates only increased the prolyl hydroxylase of the control or treated groups 20%-30%. This is reasonable because our homogenates were made in large tissue volume ratio (1: 50) and we were testing an aliquot of the entire homogenate. Initial studies demonstrated that in our hands the prolyl hydroxylase activity assayed by the procedure described above was linear with respect to time and protein content. Collagen-Bound Hepatic Hydroxyproline Synthesis and Content Mice and rats were bled with needle and syringe via their aorta under ether anesthesia. after which their livers were removed. The gallbladder of each mouse was dissected from the hepatic tissue before removal of the liver to prevent interference of hepatic function by bile acids. Livers from mice or rats were then sliced with a Stadie-Riggs microtome as described previously (30). Approximately 500 mg of liver slices were weighed and put into 25-ml Erlenmeyer flasks that contained 2.9 ml of Krebs bicarbonate buffer. and 0.4 mm proline. The mixtures were gassed for 60 s with 95% O2-5% CO2 and then 0.1 ml of buffer containing 10 flci [U)4C]proline (225 mci/ mmol. New England Nuclear Co.) was added to each flask. The flasks were sealed and incubated with shaking (100 strokes/min) at 37 C for 2 h. At the end of the reaction. the flasks were placed on ice. and 1 ml of 1.5 M sucrose was added to each flask. The mixture from each flask was homogenized (5 strokes. full speed) with a Potter-Elvehjem homogenizer. Three milliliters of the resulting homogenate from each flask was mixed with 3 ml of cold 10% TCA. The collagen-bound hydroxyproline was extracted and counted according to the method of ROjkind by using hot TCA extraction followed by chloramine-t treatment (31). In a series of initial experiments. 1 mm cycloheximide was added to inhibit protein synthesis. Under these

3 528 CARTER ET AL. GASTROENTEROLOGY Vol. 82, No.3 conditions, essentially no more radioactivity was detected in the "hydroxyproline" fraction of the flasks containing liver slices and cycloheximide than was detected iii the hydroxyproline fraction of the control flasks run as described above in the absence of any liver slices. To verify that the labeled proline was being separated from the labeled hydroxyproline, a series of recovery experiments were carried out. When [U-14C]proline was added to a liver homogenate and immediately extracted, 51% of the [U-14C]prbline radioactivity was recovered in the "proline" fraction, and 5% in the "hydroxyproline" fraction. When L-4-[G-3HJhydroxyproline (5-10 Ci/mmol, New England Nuclear Co.) was added to a liver homogenate and extracted immediately, 3% of the L-4-[G- 3 HJhydroxyproline radioactivity was recovered in the "proline" fraction, and 35% in the "hydroxyproline" fraction. These recovery levels are similar to those reported by Rojkind (31) and indicate -that the chloramine-t method yields an adequate separation of labeled hydroxyproline from labeled proline. The hot TCA extraction procedure, followed by chloramine-t treatment was also used for measuring the amount of hydroxyproline in mouse and rat liver samples. After extraction, the hydroxyproline content was analyzed colorimetrically using a-dimethylaminobenzyaldehyde (31). Lysyl Oxidase Lysyl oxidase activity in the livers of mice and rats was measured according to Siegel (32) on partially purified urea extracts after diethylaminoethyl (DEAE) treatment. One gram of liver tissue was homogenized in a glass beaker in 2 volumes of buffer (50 mm Tris-HCl, ph 7.5, 6 M urea) by using a polytron homogenizer. This homogenate Was centrifuged at 10,000 g for 15 min at 4 C, and the supernatant was removed. The supernatant was mixed at 4 C with a DEAE cellulose "cake," previously equilibrated with buffer, in a ratio of 1 g of "cake" per 2 ml of supernatant. After 30 min of stirring, the mixture was centrifuged at 10,000 g for 15 min at 4OC, and the supernatant removed. The DEAE pellet was resuspended twice with buffer and centrifuged twice at 10,000 g for 15 min at 4 C. The DEAE pellet was resuspended in buffer containing 400 mm NaCl (in order to release the lysyl oxidase), and then centrifuged at 10,000 g for 15 min at 4 C. The resulting supernatant was first passed over glass wool and then dialyzed against phosphate buffered saline (PBS). The resulting partially purified lysyl oxidase preparation (0.5 ml) was then incubated in a 5-ml plastic tube with 100 mm sodium phosphate buffer (ph 7.4), 150 mm NaCI, 10 mm lysine, and 0.3 ml of a 3H-collagen substrate containing about 1 x 10 6 cpm (pregelled for 1 h at 37 C) in a final volume of 0.9 ml. The substrate for this reaction was previously prepared from 16-day-old chick calvaria labeled with D,L-[6-3 H]lysine (>5 Ci/mmol, New England Nuclear Co.). The partially purified hepatic lysyl oxidase preparation was incubated with substrate at 37 C for 2 h with shaking (100 strokes/min) and then cooled in ice for 1 h. The 3H20 released by the lysyl oxidase reaction was distilled into counting vials as described for the measurement of prolyl hydroxylase. After the contents of the counting vials had thawed, 10 ml of Aquasol 2 (New England Nuclear Co.) was added to each of the counting vials, and the radioactivity of the contents was measured by liquid scintillation spectrometry. Activity was corrected for any counts observed in duplicate tubes run with 50 p,g of ~-aminoproprionitrile fumarate (BAPN), an inhibitor of lysyl oxidase (16,32). Lysyl oxidase activity in serum was determined as described above except that serum was used without further purification. In one set of experiments, the substrate for the lysyl oxidase reaction was a collagan preparation previously prepared from 16-day-old chick calvaria labeled with [U- 14C]lysine (225 mci/mmol, New England Nuclear Co.) (32). The partially purified hepatic lysyl oxidase preparations or the serum samples (0.5 ml) were incubated 2 h at 37 C in 10-ml glass screw-top tubes with 100 rom sodium phosphate buffer (ph 7.4), 150 mm NaCl, 10 mm lysine, and 0.3 ml of p4c]collagen substrate containing about 450,000 cpm (pregelled for 1 h at 37 C) in a final volume of 0.9 ml. The reaction was stopped by the addition of 2 ml performic acid (33) to convert the [14C]allysine (the direct reaction product of lysyl oxidase action on collagen) (33) to a-[14c]aminoadipic acid. The mixtures were neutralized with hydrobromic acid, dried down under N2, resuspended in 6 N hydrochloric acid, and hydrolyzed at 110 C for 18 h. The amino acids were separated on an amino acid analyzer (34). One-milliliter aliquots of the column fractions were added to 10 ml of Insta-Gel (Packard Instruments Co., Downers Grove, Ill.), and the radioactivity of the contents rrieasured by liquid scintillation spectrometry. Collagenase Collagenase activity was measured in mice and rat liver homogenates by using the buffer of Fujiwara et a1. (35). One gram of liver tissue was homogenized in 5 ml of 50 mm Tris-HCl (ph 7.5, 0.4 M NaCl, 5 mm CaCh, and 0.1% Triton X-I00) by using a Potter-Elvehjem homogenizer (5 strokes, full speed). The collagenase of the liverhomogenate was not activated (21) before use. Aliquots of the liver homogenate (0.1 ml) were incubated with 0.05 ml of a [U-14C]glycine (200 mci/mmol, New England Nuclear Co.) labeled guinea pig skin collagen substrate containing about 5000 cpm. The substrate had been previously prepared according to Gross (36) and pregelled 18 h at 37 C in plastic capped tubes. The collagenase reaction mixtures were incubated 18 h more at 37 C after the addition of the liver homogenates, and then centrifuged at 8800 g for 5 min at room temperature. Aliquots of the resulting supernatant (0.1 ml) containing the solubilized radioactive collagen fragments, were added to 5 ml of Insta-Gel (Packard Instruments Co.) and the radioactivity was measured by liquid scintillation spectrometry. Collagenase activity was expressed in units determined by the release of radioactivity by the liver homogenates corrected for the counts released by the blank that contained only buffer. Usually the blank contained about 4% of the original total radioactivity added. One unit of collagenase activity refers to the amount of wet weight of liver capable of degradation

4 March 1982 of 1 p.,g collagen substrate/min at 37 C, ph 7.5. In one set of experiments, after 18 h of incubation the reaction mixtures containing the liver homogenates and the [U- 14C]glycine-Iabeled substrate were treated with 5 ml of cold absolute ethanol. After standing at 4 C for 30 min, the mixtures were centrifuged at 27,000 g for 30 min. The pellets (containing the radiolabeled collagen) were dissolved in 0.5 ml of starting buffer and 0.05 ml aliquots LYSYL OXIDASE AND COLLAGENASE IN LIVER INJURY 529 electrophoresed on 7.5% polyacrylamide gels containing SDS (0.1 g/100 ml) according to Laemmli (37) with the apparatus described by Reid and Bieleski (38). Additional Tests Protein concentrations of the various liver homogenate samples were determined by the method of Lowry et Figure 1. The effect of acute and chronic liver damage on hepatic histology. A. Normal mouse liver. B.Liver from a mouse 3 days after a single dose of CCl 4 by gavage. C. Liver from a mouse 4 days after a single injection of murine hepatitis virus. D. Liver from a rat injected with CCl 4 three times weekly for a total 15 injections before death. The arrows in Band C indicate areas of cell dropout; the arrow in D indicates a fibrotic septum. (x150).

5 530 CARTER ET AL. GASTROENTEROLOGY Vol. 82, No.3 al. (39). Student's t-test was used to assess the significance of differences between the means of the various studies. Results The first series of experiments were designed to examine the effect of acute and chronic liver injury at the microscopic level. The livers taken from mice previously given either a single dose of carbon tetrachloride (CCI4 ) (Figure lb) or a single injection of murine hepatitis virus (Figure lc) showed areas of focal necrosis and cell dropout, but no fibrotic septa were evident. In contrast, chronic CCl4 treatment of rats for 6 wk resulted in the development of thick septa that stained blue with the Mallory trichrome (Figure ld). These septa outlined areas of regenerating nodules in the liver. Thus, only chronic CCl4 treatment of rats led to histologic evidence of fibrosis. The effect of acute liver injury in mice and chronic liver injury in rats on plasma GOT and various biochemical aspects of hepatic collagen deposition is shown in Table 1. The control reflects pooled data obtained from both the control mice and rats, as the values for both groups were similar. The control mice were given mineral oil by gavage or injected intraperitoneally with media containing no virus or cellular material. Control rats were injected Intraperitoneally with mineral oil. It will be seen that a single dose of carbon tetrachloride (acute CCI 4) and a sirigle injection bf muririe hepatitis virus (acute MHV-A-59) caused a marked increase (p < 0.01) in plasma GOT activity 3 and 4 days after treatment, respectively (Table n Chroriic CCl4 treatment of rats for 6 wk caused a lesser, but still significant (p < 0.01) increase in plasma GOT activity. The mean hepatic prolyl hydroxylase activity was increased two- to threefold (p < 0.05) in all three of the treated groups (acute CCI4, acute MHV-A-59, chi;onic CCI 4) as compared with control (Table 1). The synthesis of hepatic collagen-bound hydroxyproline (as measured by the incorporation of radioactivity into collagen-bound hydroxyproline) was increased twq- to fourfold (p < 0.01) in all of the treated groups (acute CCLi, acute MHV-A-59, chronic CCI 4) as compared with the controi (Table 1). Only in the chronic CCl 4-treated group was the content of hepatic collagencbound hydroxyproline significantiy increased fivefold (p < 0.01). Similarly, hepatic lysyl oxidase and hepatic collagenase activities were increased 15-fqld and fivefold, respectively (p < 0.01), only in the chronie CCl4-treated group. We next examined the effect in mice of a single dose of CCl 4 or a single injection of murine hepatitis virus on plasma GOT actlvity and various biochemical aspects of hepatic collagen deposition during a 10-day period following either initial treatment. As can be seen in Figure 2A, a single dose of CCl4 caused a maximum rise (p < 0.01) in plasma GOT activity 2 days after treatment; after a single injection of murine hepatitis virus, a maxirilum rise (p < 0.01) occurred in 4 days. As seen in Figure 2B, the maximum increases (p < o.oi) in the synthesis of collagen-bound hepatic hydroxyproline occurred 3-4 days after a single dose of CCI 4, and 4-7 days after a single injection of the virus. Hepatic lysyl oxidase or hepatic collagenase activity did not appear to be altered at any of the several time points tested (Figures 2C and 2D). As Siegel et al. (16) reported that serum lysyl oxidase activity was elevated in a fibrotic rat model, Table 1. The Effect of Acute and Chronic Liver Injury on Plasma GOT and Various Biochemical Aspects of Hepatic Collagen Deposition Control Acute CCl4 Acute MHV-A-59 Chronic CCl4 (mice & rats) (mice) (mice) (rats) (n = 33)0 (n = 10) (n = 10) (n = 3) Plasma GOTb 85 ± 10 i 6630 ± ± ± 150 Hepatic prolyl hydroxylase" 40 ± ± ± ± 15 Hepatic hydroxyproline (collagen-bound) 1. Synthesisd 4760 ± ,970 ± ± ,190 ± Content" 13 ± 4 18 ± 5 17 ± 5 75 ± 5 h Hepatic lysyl oxidasei' 58 ± ± ± ± ls0h Hepatic collagenase g 0.08 ± ± ± ± 0.09h On indicates the number of mice and rats used. As described in Methods, mice were given a. single dose of CCl4 (acute CCl 4 ) 3 days before death, or a single injection of murine hepatitis virus (acute MHV-A-59) 4 days before death. Rats were given i5 injections of CCl4 three times a week. The rats were killed 2 days after the last injectiori (chronic CCl4). b International units per ml plasma." cpm of 3H 20/ mg protein' 1 h. d cpni of [14C]hydroxyproline/g wet wt liver slices 2 h. e JLmoles hydroxyproline/g wet wt liver. f cpm of 3H 20/mg protein' 2 h. g imits/g wet wt liver. h p < 0.01, as compared to control and acutely treated mice. i Results are mean ± SD.

6 March 1982 LYSYL OXIDASE AND COLLAGENASE IN LIVER INJURY International Units/ o.01ml Plasma 8 (x 10-3) 4 A. PLASMA GOT 0--0 CCl 4 0--<) CCI 4 Control -- MHV-A MHV-A-59 Control CPM/q uel WI Tissue/ 2hrs (x 10-.3; B.HYOROXYPROLINE SYNTHESIS O~~~~=t~~~~ 100fC.LYsn OXIDASE CPM~OAng-;~ ': :.. : : : :f1 Figure 2. The effect of a single dose of CC14 or a single injection of murine hepatitis virus (MHV A-59) on mouse plasma GOT activity and various biochemical aspects of hepatic collagen metabolism. Mice were treated on day 0 with either CC1 4 or murine hepatitis virus as described in Methods and killed on the day indicated. Assay conditions are described in Methods. Three mice were used for each time point, and the mean level was plotted COLLAGENASE Units/q Wet Wt Tissue 25'~5iO-;;;';;-C:i-iiii:S ::: ~iiiiol:li==:::::::;;~~-~~~ ~ ~ ~ ~ It)... e:: I() c:s "- C) ;t::ti..i f\o) ~ Cl '-.l 0 I I I I NORMAL PARTIAL ACUTE CHRONIC HEPATECTOMY CCI4 CCI4 (24 Hrs) (3 Days) (6Weeks) Figure 3. Lysyl oxidase activity in rat serum. Assay conditions were as described in Methods, except that the enzyme source was 0.5 ml of sera obtained from the rats in each group. we examined the sera of rats with acute and chronic liver injury for the presence of lysyl oxidase activity. Rats were used instead of mice for the acute liver injury model because of the need for larger volumes of serum to measure lysyl oxidase. As seen in Figure 3. lysyl oxidase activity of rat serum was not elevated during the regenerative phase after partial hepatectomy. or 3 days after a single dose of CCI4. However. serum lysyl oxidase activity in the chronic CCl4-treated rats (6 wk) with fibrosis was markedly higher (p < 0.05) than that observed in the normal rat sera of rats with acute liver injury (Figure 3). Further studies were conducted to verify that lysyl oxidase activity was actually being measured in fibrotic rat liver and serum. Lysyl oxidase converts collagen-bound lysine and hydroxylysine to a-aminoadipic-8-semialdehyde. commonly referred to as allysine (32). It is possible to hydrolyze allysine to a aminoadipic acid that can be detected on an amino acid analyzer. When lysyl oxidase is incubated with a [14C]lysine-Iabeled collagen. and the collagen subsequently subjected to hydrolysis and amino acid analysis. it is possible to document the formation of a-[14c]aminoadipic acid (33).

7 532 CARTER ET AL. GASTROENTEROLOGY Vol. 82, No. 3 Table 2. Formation of a-[14c]aminoadipic Acid from a [14C]Lysine-Labeled Collagen by Partially Purified Hepatic Lysyl Oxidaseo and Serum Incubation with [14C]lysinelabeled collagen Partially purified hepatic lysyl oxidase Partially purified hepatic lysyl oxidase + BAPN Serum Serum + BAPN a-[14c]aminoadipic acid formation b c (total cpm) a Hepatic lysyl oxidase was purified as described in Methods. b Assay conditions for this experiment were as described in Methods. The same number of counts obtained after hydrolysis in 6 N hydrochloric acid were placed on the amino acid column. The location of the a-[14c]aminoadipic acid was determined by running a-[12c]aminoadipic acid alone through the column and then analyzing the fractions colorimetrically with ninhydrin (33,34) The results are the mean of two experiments. c The total radioactivity for the a-[13c]aminoadipic acid fraction was calculated by multiplying the cpm found in the 1 ml of the column fraction (corrected for the 33 cpm in the buffer blank) times the total volume of the fraction (2.67 ml). As seen in Table 2, a-[14c]aminoadipic acid was detected in the amino acid chromatogram of both partially purified hepatic lysyl oxidase and serum. The amount of a-[14c]aminoadipic acid produced by partially purified hepatic lysyl oxidase and serum was reduced when BAPN, an inhibitor of lysyl oxidase activity, was included in the original assay mixture. Studies were also conducted to verify that collagenase activity was actually being measured in fibrotic rat liver. When skin collagen is subjected to polyacrylamide gel electrophoresis at acid ph, two components, designated f3 and a, are observed. If skin collagen is first incubated with mammalian collagenase, and then subjected to polyacrylamide gel electrophoresis, three new faster-moving bands (designated f3a, aa, ab) are detected in addition to the intact f3 and a components (36). The products of the reaction of the guinea pig skin collagen with the fibrotic liver homogenate were detected on polyacrylamide gel electrophoresis (Figure 4). It should be noted that the substrate for this collagenase reaction was predominantly type I collagen. The electrophoretic pattern of the collagen substrate and liver homogenate (Figure 4B) contained bands (f3a, aa, ab) in addition to the f3 and a bands. No such bands were observed when the collagen substrate (Figure 4A) or the homogenate (Figure 4C) were run separately under the same conditions. Additional bands in the aa, ab regions of Figure 4B could be due to the presence of products of the collagenase action of general proteases (21). However, in data not shown, no bands other than the f3 and a components were observed when the liver homogenates were incubated with the collagen substrate in the presence of 10 mm EDTA, an inhibitor of collagenase (36). These data suggest that authentic lysyl oxidase activity was being measured in the liver and serum samples, and authentic collagenase activity was being measured in the liver samples of the chronic CCl4-treated rats with fibrosis ~ a- A a- B a- F_ A B c Figure 4. SDS-polyacrylamide gel (7.5%) electrophoretic pattern of guinea pig skin collagen degraded by chronic CCI4- treated rat liver homogenate. Assay conditions for this experiment and subsequent Coomasie blue staining of the gel were as described in the Methods section. The pattern on the left (A) is that of the collagen substrate alone; the pattern in the middle (B) is that of the collagen substrate and the liver homogenate after 18 h of incubation; the pattern on the right is that of the homogenate alone (C). The designation of a refers to single collagen chains while f3 refers to the dimers of a chains. f3a, aa and all refer to the products of the cleavage of f3 and a components. F indicates the buffer front.

8 March 1982 L YSYL OXIDASE AND COLLAGENASE IN LIVER INJURY 533 Discussion Cirrhosis is characterized by the replacement of normal liver tissue with scar tissue that is enriched with collagen. Although the rate-limiting step or steps in normal or abnormal collagen deposition are still unclear (14,15), it is known that the collagen must be initially degraded by collagenase (40) before it can be removed. Hence, a number of investigators have measured collagenase activity in normal and fibrotic livers. Increased hepatic collagenase activity has been reported in the CCl 4-treated fibrotic rat model (1,17-20) and in mice infected with Schistosoma mansoni (21). Okazaki et al. (22) have reported that chronic ethanol feeding to rats stimulated hepatic collagenase activity. Sakai et al. (24) measured the activity of an enzyme capable of digesting a short synthetic collagen substrate and found that in the fibrotic livers of rats and humans, the activity of this collagenase was increased (24). Montfront and Perez-Tamayo (25) prepared an antibody to rat uterus collagenase and used this reagent to localize collagenase in the liver capsule, in the connective tissue of the portal spaces, and along the wall of the sinusoidal capillaries in the liver parenchyma. These same authors also looked for the presence of collagenase in the livers of rats treated with CCI 4 Interestingly enough, although collagenase was detected in the liver during the reversible phase of the fibrosis, it was not detected in the livers with irreversible fibrosis (26). The results of the present study together with reports of other investigators summarized above (1,17-26) suggest that the liver normally contains a small amount of collagenase activity, and that this activity increases during some phase of hepatic fibrosis. Yet it is not clear why there appears to be an increase in collagen deposition in fibrotic livers if collagenase is increased in activity. Increased collagen deposition in the liver could result from at least four mechanisms: First, it might occur if the rate of synthesis of collagen exceeds the rate of degradation. Accurate measurements of collagen turnover in the normal and fibrotic liver, however, are currently not available. Second, increased collagen deposition might result from increased formation of type IV collagen, which is resistant to hepatic collagenase (this laboratory, unpublished observations). A number of investigators have observed that the fibrotic human liver contains more type IV collagen than normal liver (12,13). Third, an increase in collagen cross-links might occur thus rendering the collagen more resistant to collagenase. Collagen that has been crossed-linked by lysyl oxidase has been shown to be ten times more resistant to digestion by mammalian collagenase (42). In the present study and in a previous report (16) increased hepatic lysyl oxidase activity was found in the fibrotic rat liver. Fourth, increased hepatic collagen deposition might also result in part from alterations in the action of collagenase. Collagenase activity might initially be enhanced in response to increased hepatic collagen; on the other hand either the type of collagen formed or the presence of inhibitors (43) could modify the action of collagenase. In fact, it is possible that increased collagenase activity might aggravate the situation further by providing breakdown products that would stimulate disordered collagen resynthesis and thickening. Such a mechanism has been postulated as occurring in idiopathic pulmonary fibrosis (44). The present results have shown that lysyl oxidase and collagenase activities were increased in chronic CCl 4 liver injury, but were not increased in two models of acute liver injury. We have confirmed previous reports of increased hepatic prolyl hydroxylase activity in association with both acute or chronic liver injury (1-5). However, we found that only in rats with chronic CCl 4 liver injury did there ~ppear to be a correlation between hepatic fibrosis, Increased collagen-bound hydroxyproline content, increased hepatic lysyl oxidase and collagenase activity, and an increase in serum lysyl oxidase activity. Our findings suggest that lysyl oxidase and collagenase may play an important role in the collagen accumulation associated with hepatic fibrosis. References 1. Rojkind M, Dunn MA. Hepatic fibrosis. Gastroenterology 1979;76: Hirayama C. Hepatic fibrosis: biochemical considerations. In: Schaffner F, Shenker KS, eds. The liver and its diseases. Basel: Intercontinental Medical Book Corp., 1974: McGee J O'D, Fallon A. Hepatic cirrhosis-a collagen formative disease? J Clin PathoI1974;31(SuppI12): Popper H, Piez KA. Collagen metabolism in the liver. An annotated and supplemented report of a workshop at the National Institutes of Health on February 28 and March 1, Dig Dis 1978;23: Stern R. 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