Hepatotoxicity of salicylates in monolayer cell cultures

00165085/78/0074-00o26o2.oo10 h3TROENTEROLOGY 74:205-208, 1978 Copyright0 1978by the AmericanGastroenterological Association

HEPATOTOXICITY CULTURES

Vol. 74, No. 2, Part1 Printed in U.S.A.

OF SALICYLATES

KEITH G. TOLMAN, M.D., SAMUEL P. HAMMAR,

F.A.C.P.,

IN MONOLAYER CELL P. PETERSON, M.D.,

P. GRAY, B.S., AND

M.D.

Division of Gastroenterology, University of Utah College of Medicine, of Pathology, Virginia Mason Medical Center, Seattle, Washington

Salt Lake City, Utah, and Department

The influence of graded doses of sodium salicylate on rat liver cells cultured in monolayers was assessed by measuring lactic dehydrogenase activity in a culture media after incubation. Morphological alterations were studied by electron microscopy. The influence of different albumin concentrations in the media on toxicity was also evaluated. Lactic dehydrogenase concentrations rose with increasing doses of salicylate up to 40 mg per dl. High concentrations of albumin were associated with reduced salicylate toxicity. These findings suggest that salicylate-induced hepatic injury is dose related and may be influenced by serum albumin levels. Chronic high dosage salicylate therapy is of central importance in the management of patients with arthritis because of its efficacy and the relative infrequency of serious adverse reactions. Certain pathophysiological processes, specifically alterations in platelet function,2 prolongation of bleeding time,3,4 and erosion of gastric mucosa,5 are recognized as concomitants of aspirin ingestion. It now appears that hepatic injury is also directly related to salicylate therapy. This implication was first made by Manso and his colleagues6 in 1955 but it was not until 1971 that active interest in this area developed. Russell et aL7 reported elevated transaminase levels in more than 50% of a group of patients with juvenile rheumatoid arthritis and taking acetylsalicyclic acid in doses sufficient to yield blood levels higher than 35 mg per dl. “Aspirin hepatitis” has now been reported in a number of entities, including rheumatic fever, systemic lupus erythematosus, and rheumatoid arthritis.“” The common denominator in all cases is salicylate therapy, whether it is acetylsalicylic acid, sodium salicylate, or choline salicylate.12*I53l7 Elevated serum transaminase levels typically occur with serum salicylate levels of 25 mg per d112or greater, although they have been observed at salicylate levels as low as 7,11 9,g 11,15and 18 mg per d1.8 There has remained, however, some question as to the exact role of salicylates in causing the hepatic abnormalities because each of the disease states can have liver involvement within the spectrum of clinical findings. Because of this uncertainty this study was undertaken to deter-

mine the effect of salicylates on isolated rat hepatocytes in monolayer cell cultures. Because the diseases associated with hepatic injury ascribed to salicylate therapy are frequently associated with hypoalbuminemia, we also studied the influence of albumin in different concentrations on salicylate-induced alterations in hepatocyte. Materials and Methods Adult male Sprague-Dawleyrats were subjectedto a twothirds hepatectomy (median and left lateral lobes) under ether anesthesia and were permitted to recover with free access to fbod and water. Four days later the animals were used for preparationof isolatedparenchymalcells from the regeneratedliver. The liver was perfused by the method of Beny and Friendlsand the cells were isolated by a minor modificationof the methodof B&sellet al.lgHepaticperfusion was performedusing crude collagenase (Worthington Biochemical Corporation,Freehold, N. J.) in phosphatebuffer withpH adjusted to 7.4. Eagle’s minimum essential medium (MicrobiologicalAssociates,Bethesda,Md.) was used instead

of L-15 medium; gentamicin (Schering Corporation, Kenilworth, N. J.), 0.05 pg per 100 ml, was used instead of penicillin in the culture medium. Suspensions of 3.0 x 106 cells adjusted to a volume of 2.5 ml of Eagle’s minimum essential medium were placed in 60 by 15 mm contour plastic petri dishes and incubated in a humidified incubator at 37°C under 95% 0, and 5% CO,. Visual inspection using an inverted microscope was used to confirm that the rounded cells were in a continuous monolayer during all phases of the study. Exclusion of trypan blue (0.4%) was used to measure viability of cells. A complete change of medium was made after the first 24 hr of incubation. Toxicity studies. The cells were used for study after 24 hr ReceivedJanuary24, 1977. Accepted September 1, 1977. An abstract of preliminary data from this study was presented at of incubation at which time the media were poured off and the Annual Meeting of the American Federation for Clinical Re- replaced by identical media or media containing sodium salicylate (mol wt = 160.11). More than 90% of cells were search, Western Section.’ Address requests for reprints to: Dr. K. G. Tolman, Division of viable immediately after this media change. The control and Gastroenterology, Room 43504, University of Utah College of Medi- salicylate-treated cells were incubated under the usual conditions for 1, 4, 8, and 24 hr. The pH of the media was cine, 50 North Medical Drive, Salt Lake City, Utah 84132. This study was supported in part by Public Health Services maintained at 1.4. After incubation the media were withdrawn and passed through 1.2 p Millipore filters. The filtrates Grant RR-64from the Division of ResearchResources. 205

206

TOLMAN

were analyzed immediately for lactic dehydrogenase (LDH) activity using the method of Wroslewski and LaDuc.2n These values were expressed as percentage of change over control and plotted as a time-response curve (fig. 1). It was thus established that the most rapid response occurred at 1 to 8 hr and a 4-hr incubation time was chosen for the remaining

FIG. 1. Mean release of lactic dehydrogenase (LDH) is plotted as percentage change from control versus time after incubation with 40 mg of sodium salicylate per dl. The brackets represent standard

error of the mean. 50 5 B

2Ia

z

1

3.0 x 10sCELLS 4 HOUR INCUBATION

I

x 5

30-

ET AL.

studies. LDH concentration has been shown in similar in vitro studies to correlate with cell membrane disruption and is thus regarded as a good marker of cell damage and/or death under the conditions of this study.” The addition of salicylate to serum in concentrations of 10 to 50 mg per dl did not change the LDH values in the serum samples. A dose-response relationship for salicylate toxicity was established by adding salicylate in concentrations of 10, 20, 30, 40, and 50 mg per dl of incubation media. The cells were washed, then treated with salicylate and incubated for 4 hr. LDH concentration in the media was measured at 4 hr and expressed as percentage of difference from the control values with the base line adjusted to zero. Two matched control experiments were used. In the first, the media were changed but no salicylate was added. In the second, sodium bromosulfophthalein (BSP) in concentrations of 5 and 50 mg per 100 ml of media was added instead of sodium salicylate. This was done to assure that culture manipulation was not responsible for the changes in LDH concentration. In a second set of experiments, bovine albumin (fraction V, 96 to 99% albumin, Sigma Chemical Company, St. Louis, MO.) in concentrations of 1, 2, 3, 4, and 5 g per dl was added to the control and salicylate-containing media. LDH was measured after 4 hr of incubation. Morphological studies. Cells from the culture plates were washed with normal saline, fixed in Kernovsky’s solutionz2 containing phosphate buffer and 2.5% glutaraldehyde, postfixed in 2% osmium tetroxide, dehydrated in graded alcohols, and embedded in Epon 812 (Fisher Scientific Company, Fairlawn, N. J.) according to the method of Luft.23 Sections were examined with an RCA EM U4 electron microscope (RCA, Harrison, N. J.) by a microscopist without knowledge of the treatments.

Results

E 8 =

1

8 20x _I i

I

IO-

s 5 6

.

1 IO

, I 20 30 Na SALICYLATE (mQ/t

FIG. 2. Mean release of lactic dehydrogenase (LDH) is plotted as percentage change from control versus concentration of sodium salicylate in the culture media. The brackets represent standard error of the mean.

r!

2 9

Vol. 74, No. 2, Part 1

The influence of salicylate alone on LDH concentra tions in the culture media is illustrated in figure 2. The concentration of LDH increased directly with the increase in salicylate dose (r = 0.929). The increment in LDH between the salicylate doses of 30 to 40 appeared to be larger than between lower doses. Although we regarded this as artifactorial it is conceivable that the large LDH increment resulted from accelerated toxicity at the 40-mg dose. Trypan blue staining showed uptake of the dye with some disruption of the cell membranes in numbers that correlated grossly with the changes

3.0 I IO6 CELLS 4 HOUR INCUBATION . p<.Ol

l

z+40 -z 50 1

20 Na SALICYLATE

30 (mp/dl)

FIG. 3. Mean release of lactic dehydrogenase (UN-I) is plotted as percentage change from control for differing concentrations and sodium salicylate in the media. The brackets represent standard error of the mean.

of albumin

FIG. 4. Typical morphological manifestations of salicylate toxicity in cultured hepatocytes (top). Note mitochondrial swelling, disrup Ition ofthei nner mitochondrial membrane, loss of mitochondrial cristae, vacuolization, and lipid accumulation. The concentration of salicy date in this case was 40 mg per dl. Control cells (bottom) exhibited some vacuolization and lipid accumulation. 207

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TOLMAN

observed in LDH concentration. No changes were seen with BSP treatment. The influence of various concentrations of albumin on salicylate toxicity is illustrated in figure 3. At albumin concentrations of 5 g per dl there was significantly less hepatotoxicity (P < 0.01) for all concentrations of salicylate. At albumin concentrations of 4 g per dl there was a significant reduction in hepatotoxicity of salicylate concentrations of 40 and 50 mg per dl. Hypoalbuminemia levels of 1, 2, and 3 g per dl of albumin were associated with significantly more hepatotoxicity than was observed at physiological albumin levels at all sahcylate concentrations. The typical morphological manifestations of toxicity are illustrated in figure 4. The mitochondria exhibited swelling, disruption of the inner membrane, loss of cristae and clumping or decrease in matrix granules. Vacuolization and increase in cellular lipid were observed but were also found occasionally in control cells.

REFERENCES 1. Tolman KG, Peterson, P, Gray P, et al: Hepatotoxicity of

2. 3.

4.

5. 6.

7. 8. 9.

Discussion These data demonstrate that salicylate is hepatotoxic under the conditions of this study. The toxicity is dose related and influenced by the addition of albumin to the cell culture medium. The effect of hypoalbuminemia is not easily explained in view of limited information regarding the hepatic uptake of salicylate and the influence of protein binding on the uptake process. Salicylate in therapeutic concentrations in serum has been shown to be 86% bound to albumin. The rate of passive diffusion of salicylate into hepatocytes would be expected to increase if the concentration of unbound salicylate increased. Thus a reduction of albumin available for salicylate binding could result in increased hepatic uptake of salicylate and presumably increase toxicity. However, protein binding does not prevent substantial hepatic uptake of compounds that are actively transported into liver cells such as BSP. Because it is not known whether salicylate is actively or passively transported into liver cells and intracellular salicylate concentrations were not measured, we can draw no conclusions regarding the influence of albumin on hepatic uptake of salicylate in these studies. Regardless of the reason, low albumin concentrations were associated with evidence of increased salicylateinduced injury in this system. Several investigators have postulated that patients with rheumatoid arthritis, rheumatic fever, and systemic lupus erythematosus are more susceptible to salicylate hepatotoxicity. It might also be postulated that the hypoalbuminemia often associated with these disorders may contribute to the increased salicylate hepatotoxicity.

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10. 11.

12.

13. 14. 15. 16. 17.

18. 19.

20. 21.

22.

23.

salicylates in monolayer cell cultures (abstr). Clin Res 24:134A, 1976 O’Brien JR Effects of salicylates on human platelets. Lancet 1:779-783,1968 Mielke CH Jr, Kaneshiro MM, Maher J, et al: The standardized normal Ivy bleeding time and its prolongation by aspirin. Blood 34204-215, 1969 Kaneshiro, MM, Mielke CH Jr, Kasper CK, et al: Bleeding time after aspirin in disorders of intrinsic clotting. N Engl J Med 281:1039-1042, 1969 Davenport I-IWzDamage to the gastric mucosa: effects of salicylates and stimulation. Gastroenterology 49189-196, 1965 Manso C, Taranta A, Nydick I: Effect of aspirin administration on serum glutamic oxaloacetic and glutamic pyruvic transaminases in children. Proc Sot Exp Biol Med 93:83-88,1956 Russell AS, Sturge RA, Smith MA Serum transaminases during salicylate therapy. Br Med J 2:428429,1971 Athreya BH, Gorske AL, Myers AR: Aspirin-induced abnormalities of liver function. Am J Dis Child 126638-641, 1973 Seaman WE, Ishak KG, Plota PH: Aspirin-induced hepatotoxicity in patients with systemic lupus erythematosus. Ann Intern Med 8&l-8,1974 Wolfe JD, Metzger AL, Goldstein RC: Aspirin hepatitis. Ann Intern Med 80~74-76,1974 Athreya BH, Moser G, Cecil HS, et al: Aspirin-induced hepatotoxicity in juvenile rheumatoid arthritis. A prospective study. Arthritis Rheum 18:347-352, 1975 Rich RR, and Johnson JS: Salicylate hepatotoxicity in patients with juvenile rheumatoid arthritis. Arthritis Rheum 16:1-g, 1973 Garber E, Craig RM, Bahu RM: Letter: Aspirin hepatotoxicity. Ann Intern Med 82:592-593,1975 Iancu T: Letter: Serum transaminases and salicylate therapy. Br Med J 2167, 1972 Goldenberg DL: Letter: Aspirin hepatotoxicity. Ann Intern Med 80:773,1974 Koppes GM, Amett FC: Salicylate hepatotoxicity. Postgrad Med 56:193-195,1974 Drivsholm AA, Madsen S: The influence of treatment and sodium salicylate on the serum glutamic oxaloacetic transaminase activity. Stand J Clin Lab Invest 13:442446, 1961 Berry MN, Friend DS: High-yield preparation of isolated rat liver parenchymal cells. J Cell Biol43:506-520, 1969 Bissell DM, Hammaker LE, Meyer UA: Parenchymal cells from adult rat liver in nonproliferating monolayer culture. I. Functional studies. J Cell Biol59:722-731, 1973 Wroblewski F, LaDue JS: Lactic dehydrogenase activity in blood. Proc Sot Exp Biol Med 90:210-213, 1955 Dujovne CA, Shoeman D, Bianchine J, et al: Experimental bases for the different hepatotoxicity of erythromycin preparations in man. J Lab Clin Med 79832-844, 1972 Karnovsky MJ: A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy (abstr). J Cell Biol 27:137A-138A, 1965 Luft J: Improvements in epoxyresin embedding methods. J Biophys Biochem Cytol9409-414, 1961