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Preventive effects of 5-fluorouridine and uridine on d -galactosamine-induced liver injury
EXPERIMENTAL
AND
MOLECULAR
PATHOLOGY
34, 170-182 (1981)
Preventive Effects of 5Fluorouridine and Uridine D-Galactosamine-Induced Liver Injury’,* A. HOLSTEGE,~T. ANUKARAHANONTA,~,~ M. YAMAMOTO,~*~ H.K. R. KATTERMANN,~ R. LE.scH,~,~ AND D. KEPPLER~
on KOCH,~
3Biochemisches Institut der Universitiit, Hermann-Herder-S&. 7, D-7800 Freiburg, Vathologisches Institut der Universitiit, D-7800 Freiburg, and SKlinisch-Chemisches Institut, Klinikum Mannheim der Universitiit Heidelberg, D-6800 Mannheim, Federal Republic of Germany Received September 19, 1980 S-Fluorouridine, in doses of 3.5 mmole/kg body wt, was injected 3 and 6 hr after Dgalactosamine (1.85 mmole/kg) and the preventive effect on the liver injury was studied in comparison with equimolar doses of uridine in rats. The hepatic pools of UTP, UDP-glucase, and of other uracil nucleotides that were depleted by D-galactosamine were effectively replenished by formation of the respective S-fluorouracil nucleotides. 5-Fluorouridine had a similar hepatoprotective effect as uridine when studied 12 or 24 hr after administration of D-galactosamine by measurement of four different enzyme activities in plasma as well as by light and electron microscopy of the liver tissue. S-Fluorouridine was at least as effective as uridine in restoring the galactosamine-induced fragmentation of nucleoli indicating that transcription of preribosomal RNA, but not its complete maturation leading to new ribosomes, is a prerequisite for the intact nucleolar structure. In contrast to uridine, 5-fluorouridine was ineffective in supporting the replenishment of hepatic glycogen stores depleted by D-galactosamine. S-Fluorouridine and D-galactosamine had an additive effect on the reduction of lipoproteins, phospholipids, and cholesterol in plasma and on the increase of hepatic fat, whereas uridine counteracted these changes. The replacement of uridine by 5-fluorouridine in the prevention of galactosamine-induced cell necrosis allows to discriminate between those metabolic lesions that are reversed by 5-fluorouridine as well as uridine, and may be involved in the sequence of events leading to hepatocellular necrosis, and those lesions that are reversed only by uridine but not by 5-fluorouridine and are therefore not critical for hepatocellular viability within at least 24 hr.
INTRODUCTION Galactosamine (GalN)9-induced liver injury is initiated by an early depletion of hepatic UTP and UDP-hexose pools (Decker and Keppler, 1972, 1974). The consecutive inhibition of RNA synthesis can be reversed (Keppler er al., 1974; Konishi et al., 1974) and the subsequent hepatitis-like liver damage can be prevented by uridine (Urd) administered as late as 3 hr after GalN (Keppler, 1973). Fluorouridine (FUrd) can substitute for Urd in the replenishment of depleted UTP ’ Dedicated to Professor H. Holzer on the occasion of his sixtieth birthday. 2 Supported by grants from the Deutsche Forschungsgemeinschaft, Bonn, through Forschergruppe Lebererkrankungen, Freiburg. ’ Present address: Department of Pathobiology, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 4, Thailand. ’ Present address: Department of Pathology, Hiroshima University. School of Medicine, Hiroshima, Japan. B Present address: Pathologisches Institut der Stsdtischen Krankenanstalten, D-7750 Konstanz, FRG. ’ Abbreviations used: GalN, o-galactosamine; FUrd, 5-fluorouridine; Urd, uridine; UTP, uridine 5’-triphosphate; BUMP, sum of all acid-soluble uracil5’-nucleotides (Keppler et al., 1970); H(F)UMP, sum of acid-soluble uracil 5’-nucleotides and 5-fluorouracil (FUra) 5’-nucleotides; FUMP, 5fluorouridine 5’-monophosphate; GluDH, glutamate dehydrogenase (EC 1.4.1.2); GOT, aspaeate aminotransferase (EC 2.6.1.1); GPT, alanine aminotransferase (EC 2.6.1.2); SDH, r-iditol dehydrogenase (EC 1.1.1.14). 170 0014-4800/81/020170-13$02.00/O Copyright AU rights
@ 1981 by Academic Press, Inc. of reproduction in any form reserved.
GALACTOSAMINE
LIVER INJURY AND FLUOROURIDINE
171
pools (Holstege et al., 1978). Under this condition, FUTP is formed and serves as a substrate for FUDP-sugar synthesis (Roger and Perkins, 1960; Holstege et al., 1978) and transcription (Heidelberger, 1975) (Fig. 1). Replacement of Urd by FUrd in the reversal of the GalN-induced metabolic changes restores only part of the affected biosynthetic processes. This partial restoration of hepatocellular functions by FUrd serves as a means to discriminate between different pathways that may be involved in the pathogenesis of GalN-induced liver cell necrosis. The synthesis of mature ribosomal RNA remains blocked after sequential administration of GalN and FUrd whereas the formation of cytoplasmic messenger RNA (mRNA) is resumed, since incorporation of FUMP into RNA allows the synthesis of mRNA from heterogeneous nuclear RNA (hnRNA) but inhibits the processing of the ribosomal RNA precursor (pre-rRNA) (Fig. 2) (Wilkinson ef al., 1971; Hadjiolov and Hadjiolova, 1979). Furthermore, FUrd leads to an inhibition of thymidylate synthase (Hartmann and Heidelberger, 1961) thus interfering with a major pathway in the synthesis of DNA (Fig. 1). FUrd gives rise to the formation of various FUDP-sugars (Holstege et al., 1978) and reverses the deficiency of UDP-glucose and UDP-galactose by formation of their FUDP analogs. It is not known, however, whether FUrd restores UDP-dependent glycosylations after GalN administration since FUDP-sugars may not function as active substrates in glycoconjugate synthesis. The preventive effect of FUrd on the liver injury induced by GalN, as demonstrated in this communication, indicates that hepatocellular necrosis can not be related to an inhibited formation of mature ribosomal RNA and new ribosomes nor to further metabolic processes affected by FUrd. Urd has been shown to prevent and to reverse the GalN-induced nucleolar lesions (Shinozuka et al., 1973). It was of particular interest, therefore, to study the influence of FUrd on the reformation of fragmented nucleolar structures after GalN administration. A preliminary report on part of the results was given recently (Keppler et al., 1980). MATERIALS
AND METHODS
Animals Female Wistar rats (Ivanovas, Kisslegg, FRG), weighing 135 to 145 g and approximately 8 weeks of age, had free access to water and a carbohydrate-rich,
GalN
-GoLN-l-
0~b-
RN* t DNA
I
Gln+HCOj+ZATP
FIG. 1. Metabolic pathways of 5-fluorouridine
(FUrd), uridine (Urd), and tqalactosamine
(GalN).
172
HOLSTEGE
ET
AL.
hnRN*I~,M~) -'"RN*mw
A -+-----pre-rRNA&~-wrRN* ve-tRN*(FuMp)
FUrd-FUTP
I
-tRNA(F”,P)
\
FUDP-sugars-?-Gkycoconjugates 2. Consequences of FUTP utilization for transcription and FUDP-sugar synthesis. Processing and maturation of preribosomal RNA (pre-rRNA) is blocked after FUMP incorporation during transcription. By contrast, processing of the messenger RNA precursor (heterogeneous nuclear RNA, hnRNA) is apparently not disturbed. The interference of the modification at the 5-position of uracil of the transfer RNA precursor (pre-tRNA) (Randerath, 1979) is indicated. It has not been elucidated whether FUDP-sugars can serve as substrates in glycoconjugate synthesis. FIG.
20% protein diet (Altromin, Lage, FRG). Animal experiments were initiated between 9 and 11 AM; aqueous solutions of the respective compounds were neutralized with NaHCO, and injected intraperitoneally. The rats were anesthesized with pentobarbital(45 mg/kg body wt), blood was withdrawn from the aorta with heparinized syringes, and livers were freeze-clamped in situ. Chemicals and Enzymes
D-Galactosamine * HCl was purchased from C. Roth (Karlsruhe, FRG), 5fluorouridine was from Calbiochem-Behring (La Jolla, Calif), and uridine from E. Merck (Darmstadt, FRG). Cofactors, nucleotides and enzymes, used in the enzymatic analyses were from Boehringer-Mannheim (Mannheim, FRG) except for amyloglucosidase for glycogen hydrolysis that was obtained from E. Merck. Dosage and Injection Schedule
GalN was injected at zero time at a dose of 1.85 mmole/kg body wt. FUrd or Urd were administered at a dose of 3.5 mmole/kg each at 3 and 6 hr after GalN. Samples for analytical procedures were obtained 6, 12, and 24 hr after the dose of GalN. In groups treated with FUrd or Urd only, the animals were sacrificed 3, 9, or 21 hr after the initial dose of the pyrimidine nucleoside. Metabolite and Plasma Enzyme Activity
Determinations
UTP, UDP, UMP, UDP-glucose, and the sum of all acid-soluble uracil nucleotides (CUMP) were determined by specific enzymatic analyses (Keppler et al., 1970; Keppler, 1974). These assays include FUTP, FUDP, FUMP, and FUDP-glucose that are formed after administration of FUrd (Holstege er al., 1978). Glycogen was hydrolyzed with amyloglucosidase and the glucosyl residues were determined enzymatically (Keppler and Decker, 1974a). Plasma phospholipids were assayed calorimetrically (Zilversmit and Davis, 1950) and total cholesterol was measured with an enzymatic procedure (Allain et al., 1974). The following enzyme activities were measured in heparinized plasma at 25°C: Alanine aminotransferase (GPT) (Bergmeyer and Berm, 1974a), aspartate aminotransferase (GOT) (Bergmeyer and Berm, 1974b), sorbitol dehydrogenase (L-iditol dehydrogenase, SDH) (Gerlach and Hiby, 1974), glutamate dehydrogenase (GluDH) (Schmidt, 1974). Light and Electron Microscopic
Techniques
Routine light microscopy was performed on formaldehyde-fixed, parablast-embedded sections (3-5 pm thick). They were stained with hematoxylin-eosin,
GALACTOSAMINE
LIVER
INJURY
AND
FLUOROURIDINE
173
chromotrop anilin blue, orcein, elastic van Gieson, the PAS reaction with and without prior cr-amylase digestion, and Sudan red. For electron microscopic studies the tissue was immediately prefixed in a buffered glutaraldehyde solution (2% in cacodylate buffer, 0.1 mole/liter pH 7.35,4”C) for at least 24 hr. Prior to dehydration the material was postfixed in a buffered 1% osmium tetroxide solution for 1 hr. The specimens were embedded in epoxy resin according to standard procedures. From this material semithin sections (1 pm thick) were cut, stained with toluidine blue, and examined for a more detailed light microscopic characterization. Ultrathin sections of the same material were cut on an Ultracut Reichert microtome, stained with uranyl acetate and lead citrate, and studied with a Siemens electron microscope (Elmiskop 102). RESULTS Uridine
Reversal of Galactosamine-Induced Phosphate Deficiency by SFluorouridine and Uridine Urd has been shown to reverse the GalN-induced depletion of hepatic UTP, UDP, and UDP-glucose, and to augment total acid-soluble uracil nucleotides (1 UMP) which include the UDP amino sugars derived from GalN (Table I; Keppler and Decker, 1971). FUrd, too, led to an efficient replenishment of the depleted uracil nucleotide pools with 5fluorouracil nucleotides (Table I). The formation of large amounts of fluorinated uracil nucleotides was even more pronounced at 12 and 24 hr after GalN administration. FUrd alone also caused an accumulation of its nucleotide derivatives in liver which was reflected by an increase of C(F)UMP from 1.57 -C 0.16 in controls to 5.09 + 0.58 (SD, n = 6) mmole/kg of liver after 12 hr. This rise was due to an accumulation of 5-fluorouracil nucleotides in an amount of at least 3.5 mmole/kg comprising FUTP as a major component. Under corresponding conditions, Urd administration elevated the content of CUMP to 4.10 ? 0.54 (SD, n = 6) mmole/kg of liver. Enzymatic measurements of UDP-galactose (Keppler and Decker, 1974b) included FUDP-galactose and indicated that this sugar nucleotide increased as well after the doses of FUrd with or without prior GalN administration. The (F)UDP-glucose/(F)UDP-galactose ratios 12 hr after GalN + FUrd and GalN + Urd were 2.8 f 0.4 and 2.7 + 0.7 (SD, n = 5), respectively. Urd counteracted the GalN-induced drop of the hepatic glycogen content much better than FUrd (Table I). This may be related to different properties of UDP-glucose and FUDP-glucose as substrates for glycogen synthase. FUrd alone reduced the glycogen content to 156 rl: 31 mmole glucosyl units/kg of liver as compared to 288 + 40 and 288 $ 38 mmole/kg (SD, n = 5) in Urd-treated and control liver, respectively, when measured 21 and 18 hr after the two nucleoside injections. Preventive Effect of S-Fluorouridine and Uridine Indicated by Enzyme Activities in Plasma The rise of plasma activities observed 12 hr after GalN administration was completely prevented when FUrd was used to replenish the depleted uracil nucleotide pools (Table II). The effectiveness of FUrd in this respect was the same as the one demonstrated for Urd when both nucleosides were injected 3 and 6 hr after GalN (Keppler, 1973; Farber et al., 1973; Table II). Twenty-four hours after GalN administration, the preventive effect of FUrd was still highly significant and comparable to the protection against liver cell necrosis by Urd (Table II). By this
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TABLE I Uracil Nucleotides and Glycogen in Liver After Administration and 5Fluorouridine or Uridine Control
GalN
of Galactosamine
GalN + FUrd
GalN + Urd
mmole/kg (mean 2 SD, n = 5) UTP + UDP UMP UDP-glucose C UMP
0.28 0.04 0.34 1.41
4 f f 2
0.01 0.02 0.01 0.10
0.09 0.07 0.14 3.83
6 hr after GalN k 0.01 0.42 k 0.08a -c 0.01 0.10 2 0.05” 2 0.02 0.33 k 0.07” ? 0.19 4.89 2 0.30”
0.23 0.08 0.16 5.42
+ 2 k 2
0.01 0.02 0.02 0.43
UTP + UDP UMP UDP-glucose x UMP
0.25 0.05 0.30 1.57
k k k 2
0.05 0.01 0.04 0.16
0.22 0.05 0.12 5.86
k + 2 k
12 hr after GalN 0.07 0.51 k 0.12” 0.02 0.06 k 0.04” 0.02 0.22 f 0.04a 0.66 6.94 zt 0.53”
0.39 0.03 0.19 7.64
k k k 2
0.05 0.01 0.01 0.83
CUMP Glycogen (ghlcosyl units)
1.81 iz 0.15 288 f 38
24 hr after GalN 4.47 + 0.53 5.22 + 0.36” 34 k 12
4.48 2 0.68
68k 15
192 2 23
Note. FUrd or Urd were administered 3 and 6 hr after GalN; livers were freeze-clamped at the times after GalN indicated. The fluorinated uracil nucleotides are formed only after FUrd administration and are included in the enzymatic nucleotide analysis. C UMP designates the sum of all acid-soluble uracil 5’-nucleotides. u These values include the respective fluorinated uracil nucleotides derived from FUrd. TABLE II Enzyme Activities in Plasma after Administration of Galactosamine, 5-Fluorouridine, Control
GalN
GalN + FUrd
GalN + Urd
U/liter (Geometric mean, antilog SD-range; GFT GOT SDH GluDH
(15?27) 31 (25-39) 2 (l-4) (2:3)
GPT GOT
32 (27-43) 23 (14-36)
SDH GluDH
(2& 2 (2-3)
289 (217-383) 227 (175-296) 66 (44-99) 84 (57- 125) 612 (26C- 1438) 520 (225 - 1202) 146 (46-462) 77 (33- 179)
12 hr after GalN 31 37 (20-47) (23-59) 46 49 (38-60) (37-63) 5 3 (3-7) G-5) 4 3
(s-6)
C-6)
FUrd
Urd
n = 5 to 13)
(6-?7) 33 (23-45) 2 (l-2) 2
(2 1’-“,8) 26 (25-28) 2 (l-3) 2
G-3
(l-2)
24 hr after GalN 38 31 (19-73) (14-68) 68 47 (35- 131) (24-91)
41 (16- 105) 54 (27- 109)
(18!4ti) 22 (10-48)
(,OYl) 106 (46-243)
(6914) 9 (5- 15)
and Uridine
30 (20-46) 36 (26-48) (2:3) 2
(2-J)
Note. Blood samples were withdrawn at the times indicated after administration of GalN at 0 hr; FUrd or Urd were injected at 3 and 6 hr. The log normally distributed enzyme activities are given by their geometric mean and the range corresponding to ? 1 antilog SD. (McLean, 1975).
GALACTOSAMINE
LIVER
INJURY
AND
FLUOROURIDINE
175
time, however, the general toxicity of FUrd itself was reflected by a rise of plasma enzymes, particularly of glutamate dehydrogenase. The unproportional rise of the latter suggests an extrahepatic origin. The animals that had received the two doses of FUrd survived for at least 60 hr. Light Microscopic
Analysis
GalN administration provoked after 12 hr the typical signs of liver injury (reviewed by Decker and Keppler, 1972; Lesch et al., 1976) including single-cell and spotty-focal necroses accompanied by inflammatory infiltration and enlargement of Kupffer cells. FUrd doses injected 3 and 6 hr after GalN markedly reduced the extent of hepatocellular necrosis and intlammatory reaction. Light microscopy did not reveal fatty infiltration of hepatocytes by this time after GalN and FUrd. The preventive effect of Urd on GalN-induced injury after 12 hr was reflected by a minimal number of necrotic hepatocytes and inflammatory cells as compared to livers after treatment with GalN. According to the histologic features, FUrd was somewhat less effective as a protective agent than Urd in the dosage used in this study. PAS-positive but diastase-resistant cytoplasmic inclusions, corresponding to atypical dense bodies (Lesch et al., 1976), were observed whenever GalN was a component of the treatment. The histologic examination of livers 9 and 6 hr after treatment with Urd or FUrd did not show significant alterations as compared to the controls. The light microscopic changes 24 hr after GalN indicating liver injury correspond to those described after 12 hr, although hepatocellular necrosis, Councilman bodies, and inflammatory infiltration were more widespread. FUrd markedly suppressed the development of the liver injury and the histologic features resembled those observed 12 hr after GalN with subsequent FUrd. However, fatty infiltration became apparent 24 hr after GalN when FUrd had been administered in addition. According to light microscopy, Urd was more effective than FUrd in preventing the liver injury after 24 hr and fatty infiltration was not detectable. In animals treated with FUrd alone, 21 and 18 hr before sacrifice, signs of hepatitis were absent but a mild centrolobular parenchymal steatosis was observed. Electron
Microscopic
Studies on Nucleolar
Structure
As compared to the well-known structure of nucleoli in untreated rat liver (Fig. 3A; Shinozuka, 1972; Enzan et al., 1977), GalN caused the formation of small round nucleoli showing macrosegregation and composed of dense fibrillar components without granular elements (Fig. 4A). These nucleoli observed 6 hr after GalN often contained a cap of less dense fibrillar material and were partially surrounded by nucleolus-associated chromatin. An almost complete recovery of the nucleolonema was observed when liver samples were analyzed 6 hr after GalN and 3 hr after FUrd (Fig. 4C). Size and shape of nucleoli were in the normal range and there was neither macrosegregation nor microsegregation. When FUrd was replaced by Urd in the experimental schedule, the nucleolar recovery 6 hr after GalN was incomplete in most cells (Fig. 3C). The granular, fibrillar, and amorphous components could be clearly identified’ and were arranged in varying fashions representing a simple nucleolonema. In general, granular components were diffusely disposed in the center of the nucleolus and the fibrillar elements were scattered at the periphery of the nucleolus as knobs (Fig. 3C).
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FIG. 3. Electron micrographs of rat hepatocyte nucleoli after treatment with GalN, FUrd, and Urd. (A) Control. Well-organized nucleolus with sponge-like structure of nucleolonema and evenly distributed granular and tibrillar components (arrows). Nucleolus-associated chromatin (arrowheads) surrounds the nucleolus (x 25,000). (B) FUrd treatment 2 1 and 18 hr before sacrifice. Large bypergranular nucleolus (~24,000). (C) Embedding of liver 6 hr after GalN and 3 hr after Urd injection. Microsegregated nucleolus; knob-like accumulations of the fibpillar components (arrows); granular component is marked (g) (x 19,000). (D) Almost complete reformation of nucleolus 24 hr after GalN with Urd injections at 21 and 18 hr before liver removal (~23,000).
Twenty-four hours after GalN a considerable reformation of nucleolar structure is observed (Fig. 4B; Shinozuka et al., 1973; Enzan et al., 1977). The nucleoli were less compact than normal ones and composed of both granular and fibrillar components, occasionally a rope-like structure of the nucleolonema was observed. Microsegregation was indicated frequently by partial separation of tibrillar and granular components. Administration of FUrd 21 and 18 hr before sacrifice resulted in enlarged, compact, hypergranular nucleoli. They were clearly delimited from other parts of the nucleus by surrounding nucleolus-associated chromatin (Fig. 3B). A similar nucleolar structure was seen when the two doses of FUrd were given after GalN: 24 hr after the amino sugar the nucleoli were enlarged and sharply delimited (Fig. 4D); although the three distinct components were evident,
GALACTOSAMINE
LIVER INJURY AND FLUOROURIDINE
177
FIG. 4. Electron micrograph of hepatocellular nucleoli after treatment with GalN and FUrd. (A) Six hours after GalN injection. Macrosegregated nucleolus composed of dense flbrillar components and a cap (arrowhead) of less dense fibrillar material. Nucleolus-associated chromatin (CH) (~25,000). (B) Twenty-four hours after GalN injection. Microsegregated nucleolus; distinct separation of Ilbrillar (arrow) and granular (g) components (~26,100). (C) Six hours after GalN injection with FUrd treatment at 3 hr. The nucleolonema contains evenly distributed granular and llbrillar material (~25,000). (D) Twenty-four hours after GalN injection with FUrd treatment at 21 and 18 hr before sacrifice. Enlarged (note magnikation) hypergranular nucleolus (x 14,300).
the nucleolonema appeared compact and not prominent. Some cells showed hypergranular nucleoli with a predominance of large granular components. Twenty-four hours after GalN, when UTP deficiency had been reversed by two doses of Urd, the reformation of nucleolar structure was mostly complete (Fig. 3D). Effects of SFluorouridine and Uridine on Galactosamine-Induced Changes in Plasma Lipoproteins The abnormal pattern of plasma lipoproteins observed after GalN administration (Sabesin et al., 1975; Sirowej and Kattermann, 1978) represents an alteration that was prevented or reversed to a large extent by Urd but was intensified by
178
HOLSTEGE
ET AL.
FUrd (Fig. 5). The apparent loss of plasma lipoproteins in the electrophoresis pattern 24 hr after GalN with FUrd injected 21 and 18 hr before sacrifice (Fig. 5) was reflected in the reduction of plasma phospholipids and cholesterol to 28 and 21% of the control values, respectively (Table III). FUrd alone, in contrast to Urd, induced a marked reduction in the P-lipoprotein band corresponding to the lowdensity lipoproteins. This alteration was accompanied by a significant fall in the plasma concentrations of phospholipids and cholesterol to 56 and 42% of control, respectively (Table III). The additive effect of GalN and FUrd on phospholipids and cholesterol in plasma was already seen at 12 hr after GalN but it was much more pronounced after 24 hr. In none of the experimental groups (Table III) was there a significant change in the total plasma protein concentration as compared to the control range (55.4 ? 2.1 g/liter). DISCUSSION The pyrimidine nucleoside analog FUrd has in many respects a similar preventive effect on GalN-induced liver injury as Urd and can also be administered as late as 3 hr after the uridylate-trapping amino sugar. The two equimolar doses of both nucleosides administered in a 3-hr interval were required to replenish the uracil nucleotide pools for a sufficient time period (Keppler and Decker, 1971; Table I). As compared to the total FUrd dose used in the chemotherapy of tumor-bearing rats (Heidelberger et al., 1958; Anukarahanonta et al., 1980), this dose of FUrd (two times 3.5 mmole/kg) was more than 26-fold higher. The rats survived the two FUrd doses of 3.5 mmole/kg each for at least 60 hr and the signs of liver toxicity were very limited, in spite of the accumulation of large amounts of 5-fluorouracil nucleotides in this tissue (Table I) and in spite of the key role of liver in the catabolism of FUrd and FUra (Cooper et al., 1972). In agreement with studies in baboons infused with FUra into the hepatic artery (Czerwinski et al., 1975), steatosis was the predominant liver lesion detected by light and electron microscopic analysis and associated with a 3-fold increase in hepatic triglycerides 21 and 18 hr after FUrd treatment (data not shown). Hepatic steatosis is consistent with the decreased concentration of lipoproteins (Fig. 5) as well as phospholipid and cholesterol in plasma (Table III) that are mostly lipoprotein associated. FUrd
a
>
pre-6
>
I3
>
Origin
)
1
r---‘-b'
.7---T
-
FIG. 5. Plasma lipoprotein agarose electrophoresis. Plasma and 18 hr after injection of FUrd or Urd. Sudan black stain.
was obtained
0
24 hr after
GalN,
and 21
GALACTOSAMINE Phospholipids
TABLE III and Total Cholesterol in Plasma 24 hr after Administration 21 and 18 br after SFluorouridine or Uridine Control
Phospholipids Total cholesterol c DXerent
Lomb 49lT91
from the control
152
179
LIVER INJURY AND FLUOROURIDINE
GalN 746 2245 303 267
GdN +
GalN +
FUrd
Urd
mgME(mey+ 105 k 44’
a
of Galactosamine,
FUrd
+ SD; n = 9 to 13) 925 f 244 569 363 f 74
f 146’
208 f 530
Urd
1067 + 174 555 2 89
value by P < 0.001.
and GalN have an additive effect on the increase in hepatic fat and on the loss of some plasma lipoproteins. These two antipyrimidines, however, seem to affect different lipoproteins (Fig. 5) and may interfere with the synthesis and/or glycosylation of different apolipoproteins in liver. GalN has been shown to induce a loss of the C-type apolipoproteins in plasma (Sirowej and Kattermann, 1978). FUrd could interfere with the glycosylation of apolipoproteins if the respective FUDPsugars are inhibitory or inactive substrates. Furthermore, RNA synthesis in the presence of FUTP is capable of producing base-pair transformations (Glazer and Legraverend, 1980) and can result in translational errors (reviewed by Heidelberger, 1975) and could thereby lead to functionally defective apoproteins. In studies on the induction of liver enzyme proteins after administration of S-fluoroorotate, that also leads to an incorporation of FUMP into RNA, it was shown that serine dehydratase is synthesized but without normal enzymatic activity (Mohrenweiser and Pitot, 1974) whereas another enzyme protein, tyrosine aminotransferase, is even increased in activity (Cihak et al., 1973). Our experiments indicate that the reversal by FUrd of GalN-induced UTP and UDP-hexose deficiency restores those processes that are critical for hepatocellular viability during the initial 24 hr after GalN administration. The sequence of events leading to hepatocellular necrosis after a dose of GalN (Keppler, 1976) does therefore not include an interference with the following processes: (a) formation of mature ribosomal RNA (Wilkinson et al., 1971), (b) proper modification of tRNA at the 5-position of uracil (Randerath, 1979), (c) formation of proteins that are synthesized in a defective form after FUMP incorporation into RNA such as serine dehydratase (Mohrenweiser and Pitot, 1974), (d) the thymidylate synthase reaction that is most sensitive to inhibition by FdUMP (Hartmann and Heidelberger, 1961), (e) glycosyltransferase reactions that are affected by FUDP-sugars. FUDP-glucose seems to be an inadequate substitute for UDP-glucose in the glycogen synthase reaction as the hepatic glycogen was resynthesized only poorly ,when FUrd was injected after GalN whereas Urd was capable of restoring the liver glycogen to a large extent (Table I). Studies with isolated sugar transferases are required for a better understanding of the role of FUDP-sugars in glycoprotein and glycolipid synthesis. The GalN-induced block in transcription (Keppler et al., 1974; Konishi et al., 1974; Gajdardjieva et al., 1980) leads to extensive nucleolar fragmentation with a loss of granular components (Shinozuka, 1972; Shinozuka et al., 1473; Enzan et al., 1977; Dimova et al., 1979). This alteration is a common manifestation of ribosomal RNA synthesis inhibition by several agents (Shinozuka, 1972). The effective reversal of this nucleolar lesion by FUrd demonstrates (Fig. 4) that the
180
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ET AL.
formation of preribosomal RNA, but not its complete maturation, is the prerequisite for the reformation of an intact nucleolar structure. Although maturation of ribosomal RNA and the subsequent formation of new ribosomes remain blocked under this condition, a limited processing of the 45 S preribosomal RNA to 36 and 32 S precursor molecules can proceed (Hadjiolov and Hadjiolova, 1979). FUrd is at least as effective as Urd in the reversal of the GalN-induced nucleolar fragmentation (Figs. 3C and 4C). The hypergranular nucleoli observed 21 and 18 hr after FUrd (Figs. 3B and 4D) may reflect the accumulation of the ribosomal RNA precursors that cannot be processed further and released into the cytosol. ACKNOWLEDGMENTS We are indebted to Christa Holstege for her excellent technical assistance and to Karl Happersberger for his expert help in all animal experiments.
REFERENCES ALLAIN, C. C., POON, L. S., CHAN, C. S., RICHMOND, W., and FIJ, P. C. (1974). Enzymatic determination of total serum cholesterol. C/in. Chem. 20, 470-475. ANUKARAHANONTA, T., HOLSTEGE, A., and KEPPLER, D. (1980). Selective enhancement of 5fluorouridine uptake and action in rat hepatomas in vivo following pretreatment with n-galactosamine and 6-azauridine or N-(phosphonacetyl)-L-aspartate. Eur. J. Cancer 16, 1171- 1180. BERGMEYER, H. U., and BERNT, E. (1974a). Glutamate-pyruvate transaminase. UV-assay, manual method. In “Methods of Enzymatic Analysis” (H.U. Bergmeyer, ed.) 2nd Ed., pp. 752-758. Academic Press, New York. BERGMEYER, H. U., and BERNT, E. (1974b). Glutamate-oxalacetate transaminase. UV-assay, manual method. In “Methods of Enzymatic Analysis” (H. U. Bergmeyer, Ed.) 2nd Ed., pp. 727-733. Academic Press, New York. CIHAK, A., LAMAR, C., and PITOT, H. C. (1973). Studies on the mechanism of the stimulation of tyrosine aminotransferase activity in vivo by pyrimidine analogs: The role of enzyme synthesis and degradation. Arch. Biochem. Biophys. 156, 176-187. COOPER, G. M., DUNNING, W. F., and GREER, S. (1972). Role of catabolism in pyrimidine utilization for nucleic acid synthesis in vivo. Cancer Res. 32, 390-397. CZERWINSKI, W., DELARUE, J. C., DUBOLJCH,P., and LOISILLIER, F. (1975). Action de medicaments cytotoxiques sur le tissu hepatique normal du Babouin. Lesions immediates et tardives. Bull. Cancer (Paris) 62, 37-48. DECKER, K., and KEPPLER, D. (1972). Galactosamine-induced liver injury. In “Progress in Liver Diseases”. (H. Popper and F. Schaffner, eds.), Vol. 4, pp. 183- 199. Grune & Stratton, New York. DECKER, K., and KEPPLER, D. (1974). Galactosamine hepatitis: Key role of the nucleotide deficiency period in the pathogenesis of cell injury and cell death. Rev. Physiol. Biochem. Pharmacof. 71, 77- 106. DIMOVA, R. N., GAJDARDJIEVA, K. C., DABEVA, M. D., and HADIIOLOV, A. A. (1979). Early effects of D-galactosamine on rat liver nucleolar structures. Biol. Cdlulaire 35, 1- 10. ENZAN, H., HKBERLE, B., MEINHARDT, K., SCHUCHARDT, CH., KOCH, H. K., and LESCH, R. (1977). Adaptive changes of the rat liver cells induced by repeated intraperitoneal injections of D-galactosamine. II. Light and electron microscopic investigations of hepatocellular nucleolar alterations. Virchow’s Arch. B. Cell. Pathol. 26, 59-71. FARBER, J., GILL, F., and KONISHI, Y. (1973). Prevention of galactosamine-induced liver cell necrosis by uridine. Amer. J. Pathol. 72, 53-62. GAJDARDJIEVA, K. C., DABEVA, M. D., and HADJIOLOV, A. A. (1980). Maturation and nucleocytoplasmic transport of rat-liver ribosomal RNA upon o-galactosamine inhibition of transcription. Eur. J. Biochem. 104, 451-458. GERLACH, V., and HIBY, W. (1974). Sorbitol dehydrogenase. In “Methods of Enzymatic Analysis” (H. U. Bergmeyer, ed.) 2nd Ed., pp. 569-573. Academic Press, New York. GLAZER, R. I., and LEGRAVEREND, M. (1980). The effect of 5-fluorouridine 5’-triphosphate on RNA transcribed in isolated nuclei in vitro. Mol. Pharmacol. 17, 279-282. HAD~IOLOV, A. A., and HADJIOLOVA, K. V. (1979). The effect of 5-fluoropyrimidines on the processing of ribonucleic acids in liver. In “Antimetabolites in Biochemistry, Biology and Medicine” (J. Skoda and P. Langen, eds.), Vol. 57, pp. 77-85. FEBS-Symposia, Pergamon Press, Oxford.
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K. U., and HEIDELBERGER, C. (1961). Studies on fluorinated pyrimidines. XIII. Inhibition of thymidylate synthetase. J. Viol. Chem. 263, 3006-3013. HEIDELBERGER, C. (1975). Fluorinated pyrimidines and their nucleosides. In “Antineoplastic and Immunosuppressive Agents” (A. C. Sartorelli and D. G. Johns, eds.), Vol. 38, pp. 193-231. Springer-Verlag, Heidelberg. HEIDELBERGER, C., GRIESBACH, L., CRUZ, O., SCHNITZER, R. J., and GRUNBERG, F. (1958). Fluorinated pyrimidines. VI. Effects of S-fluorouridine and 5’-fluoro-2’-deoxyuridine on transplanted tumors. Proc. Sot. Exp. Biol. 97,470-475. HOLSTEGE, A., HERRMANN, B., and KEPPLER, D. (1978). Increased formation of nucleotide derivatives of 5-fluorouridine in hepatoma cells treated with inhibitors of pyrimidine synthesis and Dgalactosamine. FEBS Left. 95, 361-365. KEPPLER, D. (1973). Hepatite exp&imentale a la o-galactosamine. Ann. GastroentCrol. Hepatol. 9, 211-221. KEPPLER, D. (1974). Determination of 5’-nucleotides as nucleoside-5’-monophosphates. In “Methods of Enzymatic Analysis” (H. U. Bergmeyer, ed.), 2nd Ed., pp. 2088-2096. Academic Press, New York. KEPPLER, D. (1976). Ribonucleic acid synthesis inhibition and cell necrosis induced by selective uridine triphosphate deficiency in liver and ascites hepatoma cells. In “Toxic Liver Injury” (M. U. Dianzani, G. Ugazio, and L. hi. Sena, eds.), pp. 132- 138. Minerva Medica, Torino. KEPPLER, D., ANUKARAHANONTA, T., HOLSTEGE, A., KATTERMANN, R., and LESCH, R. (1980). Preventive effect of 5-fluorouridine on galactosamine-induced liver injury. Hoppe-Seyler’s 2. Physiol. Chem. 361, 281. KEPPLER, D., and DECKER, K. (1971). Der Wirkungsmechanismus von D-Galaktosamin in der Leber. Verh. Dtsch. Ges. Inn. Med. 77, 1182- 1185. KEPPLER, D., and DECKER, K. (1974a). Glycogen. Determination with amyloglucosidase. In “Methods of Enzymatic Analysis” (H. U. Bergmeyer, ed.) 2nd Ed., pp. 1127-1131. Academic Press, New York. KEPPLER, D., and DECKER, K. (1974b). UridineJ’-diphosphogalactose. In “Methods of Enzymatic Analysis” (H. U. Bergmeyer, ed.), 2nd Ed., pp. 2221-2224. Academic Press, New York. KEPPLER, D., PAUSCH, J., and DECKER, K. (1974). Selective uridine triphosphate deficiency induced by o-galactosamine in liver and reversed by pyrimidine nucleotide precursors. Effect on ribonucleic acid synthesis. .Z. Biol. Chem. 249, 21 l-216. KEPPLER, D., RUDIGIER, J., and DECKER, K. (1970). Enzymic determination of uracil nucleotides in tissues. Anal. Biochem. 38, 105- 114. KONISHI , Y., SHINOZUKA, H., and FARBER, J. L. (1974). The inhibition of rat liver nuclear ribonucleic acid synthesis by galactosamine and its reversal by uridine. Lab. Invest. 30, 751-756. LESCH, R., MEINHARDT, K., HRBERLE, B., and ENZAN, H. (1976). The appearance and degradation of speciBc hepatocellular cytoplasmic inclusion bodies in rat liver due to o-galactosamine. I. The relation between the amount of liver glycogen and the appearance of the atypical dense bodies in the liver cell. Virchow’s Arch. B. Cell Pathol. 21, 313-327. MCLEAN, A. E. M. (1975). The measurement of liver injury and protection, with special reference to paracetamol, dimethylnitrosamine and carbon tetrachloride. In “Pathogenesis and Mechanisms of Liver Cell Necrosis” (D. Keppler, ed.), pp. 119-127. MTP Press, Lancaster. MOHRENWEISER, H. W., and PITOT, H. C. (1974). Studies on the mechanism of 5-fluoroorotic acid inhibition of setine dehydratase induction. Arch. Biochem. Biophys. 164, 663-668. RANDERATH, K. (1979). Drug effects on tRNA. In “Effects of Drugs on the Cell Nucleus” (H. Busch, S. T. Crooke, and Y. Daskal, eds.), pp. 275-299. Academic Press, New York. ROGERS, H. J., and PERKINS, H. R. (1960). 5-Fluorouracil and mucopeptide biosynthesis by Staphylococcus aureus. Biochem. J. 77,448-459. SABESIN, S. M., KUIKEN, L. B., and RAGLAND, J. B. (1975). Lipoprotein and lecithimcholesterol acyltransferase changes in galactosamine-induced rat liver injury. Science 190, 1302-1304. SCHMIDT, E. (1974). Glutamate-dehydrogenase. UV-assay. In “Methods of Enzymatic Analysis” (H. U. Bergmeyer, ed.), 2nd Ed., pp. 650-656. Academic Press, New York. SHINOZUKA, H. (1972). Response of nucleus and nucleolus to inhibition of RNA synthesis. In “The Pathology of Transcription and Translation” (E. Farber, ed.), pp. 73-103. Dekker, New York. SHINOZUKA, H., MARTIN, J. T., and FARBER, T. L. (1973). The induction of fibrillar nucleoli in rat liver cells by tr-galactosamine and their subsequent re-formation into normal nucleoli. J. Ultrasfruct. Res. 44. 279-292. HARTMANN,
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SIROWEJ, H., and KATTERMANN, R. (1978). Apolipoproteins of rats treated with D-galactosamine. Hoppe-Seyler’s 2. Physiol. Chem. 359, 1443- 1445. WILKINSON, D. S., CIHAK, A., and PITOT, H. C. (1971). Inhibition of ribosomal ribonucleic acid maturation in rat liver by 5-fluoroorotic acid resulting in the selective labeling of cytoplasmic messenger t&nucleic acid. J. Biol. Chem. 246, 6418-6427. ZILVERSMIT, D. B., and DAVIS, A. K. (1950). Microdetermination of plasma phospholipids by trichloroacetic acid precipitation. J. Lab. Clin. Med. 35, 155-160.
AND
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PATHOLOGY
34, 170-182 (1981)
Preventive Effects of 5Fluorouridine and Uridine D-Galactosamine-Induced Liver Injury’,* A. HOLSTEGE,~T. ANUKARAHANONTA,~,~ M. YAMAMOTO,~*~ H.K. R. KATTERMANN,~ R. LE.scH,~,~ AND D. KEPPLER~
on KOCH,~
3Biochemisches Institut der Universitiit, Hermann-Herder-S&. 7, D-7800 Freiburg, Vathologisches Institut der Universitiit, D-7800 Freiburg, and SKlinisch-Chemisches Institut, Klinikum Mannheim der Universitiit Heidelberg, D-6800 Mannheim, Federal Republic of Germany Received September 19, 1980 S-Fluorouridine, in doses of 3.5 mmole/kg body wt, was injected 3 and 6 hr after Dgalactosamine (1.85 mmole/kg) and the preventive effect on the liver injury was studied in comparison with equimolar doses of uridine in rats. The hepatic pools of UTP, UDP-glucase, and of other uracil nucleotides that were depleted by D-galactosamine were effectively replenished by formation of the respective S-fluorouracil nucleotides. 5-Fluorouridine had a similar hepatoprotective effect as uridine when studied 12 or 24 hr after administration of D-galactosamine by measurement of four different enzyme activities in plasma as well as by light and electron microscopy of the liver tissue. S-Fluorouridine was at least as effective as uridine in restoring the galactosamine-induced fragmentation of nucleoli indicating that transcription of preribosomal RNA, but not its complete maturation leading to new ribosomes, is a prerequisite for the intact nucleolar structure. In contrast to uridine, 5-fluorouridine was ineffective in supporting the replenishment of hepatic glycogen stores depleted by D-galactosamine. S-Fluorouridine and D-galactosamine had an additive effect on the reduction of lipoproteins, phospholipids, and cholesterol in plasma and on the increase of hepatic fat, whereas uridine counteracted these changes. The replacement of uridine by 5-fluorouridine in the prevention of galactosamine-induced cell necrosis allows to discriminate between those metabolic lesions that are reversed by 5-fluorouridine as well as uridine, and may be involved in the sequence of events leading to hepatocellular necrosis, and those lesions that are reversed only by uridine but not by 5-fluorouridine and are therefore not critical for hepatocellular viability within at least 24 hr.
INTRODUCTION Galactosamine (GalN)9-induced liver injury is initiated by an early depletion of hepatic UTP and UDP-hexose pools (Decker and Keppler, 1972, 1974). The consecutive inhibition of RNA synthesis can be reversed (Keppler er al., 1974; Konishi et al., 1974) and the subsequent hepatitis-like liver damage can be prevented by uridine (Urd) administered as late as 3 hr after GalN (Keppler, 1973). Fluorouridine (FUrd) can substitute for Urd in the replenishment of depleted UTP ’ Dedicated to Professor H. Holzer on the occasion of his sixtieth birthday. 2 Supported by grants from the Deutsche Forschungsgemeinschaft, Bonn, through Forschergruppe Lebererkrankungen, Freiburg. ’ Present address: Department of Pathobiology, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 4, Thailand. ’ Present address: Department of Pathology, Hiroshima University. School of Medicine, Hiroshima, Japan. B Present address: Pathologisches Institut der Stsdtischen Krankenanstalten, D-7750 Konstanz, FRG. ’ Abbreviations used: GalN, o-galactosamine; FUrd, 5-fluorouridine; Urd, uridine; UTP, uridine 5’-triphosphate; BUMP, sum of all acid-soluble uracil5’-nucleotides (Keppler et al., 1970); H(F)UMP, sum of acid-soluble uracil 5’-nucleotides and 5-fluorouracil (FUra) 5’-nucleotides; FUMP, 5fluorouridine 5’-monophosphate; GluDH, glutamate dehydrogenase (EC 1.4.1.2); GOT, aspaeate aminotransferase (EC 2.6.1.1); GPT, alanine aminotransferase (EC 2.6.1.2); SDH, r-iditol dehydrogenase (EC 1.1.1.14). 170 0014-4800/81/020170-13$02.00/O Copyright AU rights
@ 1981 by Academic Press, Inc. of reproduction in any form reserved.
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pools (Holstege et al., 1978). Under this condition, FUTP is formed and serves as a substrate for FUDP-sugar synthesis (Roger and Perkins, 1960; Holstege et al., 1978) and transcription (Heidelberger, 1975) (Fig. 1). Replacement of Urd by FUrd in the reversal of the GalN-induced metabolic changes restores only part of the affected biosynthetic processes. This partial restoration of hepatocellular functions by FUrd serves as a means to discriminate between different pathways that may be involved in the pathogenesis of GalN-induced liver cell necrosis. The synthesis of mature ribosomal RNA remains blocked after sequential administration of GalN and FUrd whereas the formation of cytoplasmic messenger RNA (mRNA) is resumed, since incorporation of FUMP into RNA allows the synthesis of mRNA from heterogeneous nuclear RNA (hnRNA) but inhibits the processing of the ribosomal RNA precursor (pre-rRNA) (Fig. 2) (Wilkinson ef al., 1971; Hadjiolov and Hadjiolova, 1979). Furthermore, FUrd leads to an inhibition of thymidylate synthase (Hartmann and Heidelberger, 1961) thus interfering with a major pathway in the synthesis of DNA (Fig. 1). FUrd gives rise to the formation of various FUDP-sugars (Holstege et al., 1978) and reverses the deficiency of UDP-glucose and UDP-galactose by formation of their FUDP analogs. It is not known, however, whether FUrd restores UDP-dependent glycosylations after GalN administration since FUDP-sugars may not function as active substrates in glycoconjugate synthesis. The preventive effect of FUrd on the liver injury induced by GalN, as demonstrated in this communication, indicates that hepatocellular necrosis can not be related to an inhibited formation of mature ribosomal RNA and new ribosomes nor to further metabolic processes affected by FUrd. Urd has been shown to prevent and to reverse the GalN-induced nucleolar lesions (Shinozuka et al., 1973). It was of particular interest, therefore, to study the influence of FUrd on the reformation of fragmented nucleolar structures after GalN administration. A preliminary report on part of the results was given recently (Keppler et al., 1980). MATERIALS
AND METHODS
Animals Female Wistar rats (Ivanovas, Kisslegg, FRG), weighing 135 to 145 g and approximately 8 weeks of age, had free access to water and a carbohydrate-rich,
GalN
-GoLN-l-
0~b-
RN* t DNA
I
Gln+HCOj+ZATP
FIG. 1. Metabolic pathways of 5-fluorouridine
(FUrd), uridine (Urd), and tqalactosamine
(GalN).
172
HOLSTEGE
ET
AL.
hnRN*I~,M~) -'"RN*mw
A -+-----pre-rRNA&~-wrRN* ve-tRN*(FuMp)
FUrd-FUTP
I
-tRNA(F”,P)
\
FUDP-sugars-?-Gkycoconjugates 2. Consequences of FUTP utilization for transcription and FUDP-sugar synthesis. Processing and maturation of preribosomal RNA (pre-rRNA) is blocked after FUMP incorporation during transcription. By contrast, processing of the messenger RNA precursor (heterogeneous nuclear RNA, hnRNA) is apparently not disturbed. The interference of the modification at the 5-position of uracil of the transfer RNA precursor (pre-tRNA) (Randerath, 1979) is indicated. It has not been elucidated whether FUDP-sugars can serve as substrates in glycoconjugate synthesis. FIG.
20% protein diet (Altromin, Lage, FRG). Animal experiments were initiated between 9 and 11 AM; aqueous solutions of the respective compounds were neutralized with NaHCO, and injected intraperitoneally. The rats were anesthesized with pentobarbital(45 mg/kg body wt), blood was withdrawn from the aorta with heparinized syringes, and livers were freeze-clamped in situ. Chemicals and Enzymes
D-Galactosamine * HCl was purchased from C. Roth (Karlsruhe, FRG), 5fluorouridine was from Calbiochem-Behring (La Jolla, Calif), and uridine from E. Merck (Darmstadt, FRG). Cofactors, nucleotides and enzymes, used in the enzymatic analyses were from Boehringer-Mannheim (Mannheim, FRG) except for amyloglucosidase for glycogen hydrolysis that was obtained from E. Merck. Dosage and Injection Schedule
GalN was injected at zero time at a dose of 1.85 mmole/kg body wt. FUrd or Urd were administered at a dose of 3.5 mmole/kg each at 3 and 6 hr after GalN. Samples for analytical procedures were obtained 6, 12, and 24 hr after the dose of GalN. In groups treated with FUrd or Urd only, the animals were sacrificed 3, 9, or 21 hr after the initial dose of the pyrimidine nucleoside. Metabolite and Plasma Enzyme Activity
Determinations
UTP, UDP, UMP, UDP-glucose, and the sum of all acid-soluble uracil nucleotides (CUMP) were determined by specific enzymatic analyses (Keppler et al., 1970; Keppler, 1974). These assays include FUTP, FUDP, FUMP, and FUDP-glucose that are formed after administration of FUrd (Holstege er al., 1978). Glycogen was hydrolyzed with amyloglucosidase and the glucosyl residues were determined enzymatically (Keppler and Decker, 1974a). Plasma phospholipids were assayed calorimetrically (Zilversmit and Davis, 1950) and total cholesterol was measured with an enzymatic procedure (Allain et al., 1974). The following enzyme activities were measured in heparinized plasma at 25°C: Alanine aminotransferase (GPT) (Bergmeyer and Berm, 1974a), aspartate aminotransferase (GOT) (Bergmeyer and Berm, 1974b), sorbitol dehydrogenase (L-iditol dehydrogenase, SDH) (Gerlach and Hiby, 1974), glutamate dehydrogenase (GluDH) (Schmidt, 1974). Light and Electron Microscopic
Techniques
Routine light microscopy was performed on formaldehyde-fixed, parablast-embedded sections (3-5 pm thick). They were stained with hematoxylin-eosin,
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chromotrop anilin blue, orcein, elastic van Gieson, the PAS reaction with and without prior cr-amylase digestion, and Sudan red. For electron microscopic studies the tissue was immediately prefixed in a buffered glutaraldehyde solution (2% in cacodylate buffer, 0.1 mole/liter pH 7.35,4”C) for at least 24 hr. Prior to dehydration the material was postfixed in a buffered 1% osmium tetroxide solution for 1 hr. The specimens were embedded in epoxy resin according to standard procedures. From this material semithin sections (1 pm thick) were cut, stained with toluidine blue, and examined for a more detailed light microscopic characterization. Ultrathin sections of the same material were cut on an Ultracut Reichert microtome, stained with uranyl acetate and lead citrate, and studied with a Siemens electron microscope (Elmiskop 102). RESULTS Uridine
Reversal of Galactosamine-Induced Phosphate Deficiency by SFluorouridine and Uridine Urd has been shown to reverse the GalN-induced depletion of hepatic UTP, UDP, and UDP-glucose, and to augment total acid-soluble uracil nucleotides (1 UMP) which include the UDP amino sugars derived from GalN (Table I; Keppler and Decker, 1971). FUrd, too, led to an efficient replenishment of the depleted uracil nucleotide pools with 5fluorouracil nucleotides (Table I). The formation of large amounts of fluorinated uracil nucleotides was even more pronounced at 12 and 24 hr after GalN administration. FUrd alone also caused an accumulation of its nucleotide derivatives in liver which was reflected by an increase of C(F)UMP from 1.57 -C 0.16 in controls to 5.09 + 0.58 (SD, n = 6) mmole/kg of liver after 12 hr. This rise was due to an accumulation of 5-fluorouracil nucleotides in an amount of at least 3.5 mmole/kg comprising FUTP as a major component. Under corresponding conditions, Urd administration elevated the content of CUMP to 4.10 ? 0.54 (SD, n = 6) mmole/kg of liver. Enzymatic measurements of UDP-galactose (Keppler and Decker, 1974b) included FUDP-galactose and indicated that this sugar nucleotide increased as well after the doses of FUrd with or without prior GalN administration. The (F)UDP-glucose/(F)UDP-galactose ratios 12 hr after GalN + FUrd and GalN + Urd were 2.8 f 0.4 and 2.7 + 0.7 (SD, n = 5), respectively. Urd counteracted the GalN-induced drop of the hepatic glycogen content much better than FUrd (Table I). This may be related to different properties of UDP-glucose and FUDP-glucose as substrates for glycogen synthase. FUrd alone reduced the glycogen content to 156 rl: 31 mmole glucosyl units/kg of liver as compared to 288 + 40 and 288 $ 38 mmole/kg (SD, n = 5) in Urd-treated and control liver, respectively, when measured 21 and 18 hr after the two nucleoside injections. Preventive Effect of S-Fluorouridine and Uridine Indicated by Enzyme Activities in Plasma The rise of plasma activities observed 12 hr after GalN administration was completely prevented when FUrd was used to replenish the depleted uracil nucleotide pools (Table II). The effectiveness of FUrd in this respect was the same as the one demonstrated for Urd when both nucleosides were injected 3 and 6 hr after GalN (Keppler, 1973; Farber et al., 1973; Table II). Twenty-four hours after GalN administration, the preventive effect of FUrd was still highly significant and comparable to the protection against liver cell necrosis by Urd (Table II). By this
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TABLE I Uracil Nucleotides and Glycogen in Liver After Administration and 5Fluorouridine or Uridine Control
GalN
of Galactosamine
GalN + FUrd
GalN + Urd
mmole/kg (mean 2 SD, n = 5) UTP + UDP UMP UDP-glucose C UMP
0.28 0.04 0.34 1.41
4 f f 2
0.01 0.02 0.01 0.10
0.09 0.07 0.14 3.83
6 hr after GalN k 0.01 0.42 k 0.08a -c 0.01 0.10 2 0.05” 2 0.02 0.33 k 0.07” ? 0.19 4.89 2 0.30”
0.23 0.08 0.16 5.42
+ 2 k 2
0.01 0.02 0.02 0.43
UTP + UDP UMP UDP-glucose x UMP
0.25 0.05 0.30 1.57
k k k 2
0.05 0.01 0.04 0.16
0.22 0.05 0.12 5.86
k + 2 k
12 hr after GalN 0.07 0.51 k 0.12” 0.02 0.06 k 0.04” 0.02 0.22 f 0.04a 0.66 6.94 zt 0.53”
0.39 0.03 0.19 7.64
k k k 2
0.05 0.01 0.01 0.83
CUMP Glycogen (ghlcosyl units)
1.81 iz 0.15 288 f 38
24 hr after GalN 4.47 + 0.53 5.22 + 0.36” 34 k 12
4.48 2 0.68
68k 15
192 2 23
Note. FUrd or Urd were administered 3 and 6 hr after GalN; livers were freeze-clamped at the times after GalN indicated. The fluorinated uracil nucleotides are formed only after FUrd administration and are included in the enzymatic nucleotide analysis. C UMP designates the sum of all acid-soluble uracil 5’-nucleotides. u These values include the respective fluorinated uracil nucleotides derived from FUrd. TABLE II Enzyme Activities in Plasma after Administration of Galactosamine, 5-Fluorouridine, Control
GalN
GalN + FUrd
GalN + Urd
U/liter (Geometric mean, antilog SD-range; GFT GOT SDH GluDH
(15?27) 31 (25-39) 2 (l-4) (2:3)
GPT GOT
32 (27-43) 23 (14-36)
SDH GluDH
(2& 2 (2-3)
289 (217-383) 227 (175-296) 66 (44-99) 84 (57- 125) 612 (26C- 1438) 520 (225 - 1202) 146 (46-462) 77 (33- 179)
12 hr after GalN 31 37 (20-47) (23-59) 46 49 (38-60) (37-63) 5 3 (3-7) G-5) 4 3
(s-6)
C-6)
FUrd
Urd
n = 5 to 13)
(6-?7) 33 (23-45) 2 (l-2) 2
(2 1’-“,8) 26 (25-28) 2 (l-3) 2
G-3
(l-2)
24 hr after GalN 38 31 (19-73) (14-68) 68 47 (35- 131) (24-91)
41 (16- 105) 54 (27- 109)
(18!4ti) 22 (10-48)
(,OYl) 106 (46-243)
(6914) 9 (5- 15)
and Uridine
30 (20-46) 36 (26-48) (2:3) 2
(2-J)
Note. Blood samples were withdrawn at the times indicated after administration of GalN at 0 hr; FUrd or Urd were injected at 3 and 6 hr. The log normally distributed enzyme activities are given by their geometric mean and the range corresponding to ? 1 antilog SD. (McLean, 1975).
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time, however, the general toxicity of FUrd itself was reflected by a rise of plasma enzymes, particularly of glutamate dehydrogenase. The unproportional rise of the latter suggests an extrahepatic origin. The animals that had received the two doses of FUrd survived for at least 60 hr. Light Microscopic
Analysis
GalN administration provoked after 12 hr the typical signs of liver injury (reviewed by Decker and Keppler, 1972; Lesch et al., 1976) including single-cell and spotty-focal necroses accompanied by inflammatory infiltration and enlargement of Kupffer cells. FUrd doses injected 3 and 6 hr after GalN markedly reduced the extent of hepatocellular necrosis and intlammatory reaction. Light microscopy did not reveal fatty infiltration of hepatocytes by this time after GalN and FUrd. The preventive effect of Urd on GalN-induced injury after 12 hr was reflected by a minimal number of necrotic hepatocytes and inflammatory cells as compared to livers after treatment with GalN. According to the histologic features, FUrd was somewhat less effective as a protective agent than Urd in the dosage used in this study. PAS-positive but diastase-resistant cytoplasmic inclusions, corresponding to atypical dense bodies (Lesch et al., 1976), were observed whenever GalN was a component of the treatment. The histologic examination of livers 9 and 6 hr after treatment with Urd or FUrd did not show significant alterations as compared to the controls. The light microscopic changes 24 hr after GalN indicating liver injury correspond to those described after 12 hr, although hepatocellular necrosis, Councilman bodies, and inflammatory infiltration were more widespread. FUrd markedly suppressed the development of the liver injury and the histologic features resembled those observed 12 hr after GalN with subsequent FUrd. However, fatty infiltration became apparent 24 hr after GalN when FUrd had been administered in addition. According to light microscopy, Urd was more effective than FUrd in preventing the liver injury after 24 hr and fatty infiltration was not detectable. In animals treated with FUrd alone, 21 and 18 hr before sacrifice, signs of hepatitis were absent but a mild centrolobular parenchymal steatosis was observed. Electron
Microscopic
Studies on Nucleolar
Structure
As compared to the well-known structure of nucleoli in untreated rat liver (Fig. 3A; Shinozuka, 1972; Enzan et al., 1977), GalN caused the formation of small round nucleoli showing macrosegregation and composed of dense fibrillar components without granular elements (Fig. 4A). These nucleoli observed 6 hr after GalN often contained a cap of less dense fibrillar material and were partially surrounded by nucleolus-associated chromatin. An almost complete recovery of the nucleolonema was observed when liver samples were analyzed 6 hr after GalN and 3 hr after FUrd (Fig. 4C). Size and shape of nucleoli were in the normal range and there was neither macrosegregation nor microsegregation. When FUrd was replaced by Urd in the experimental schedule, the nucleolar recovery 6 hr after GalN was incomplete in most cells (Fig. 3C). The granular, fibrillar, and amorphous components could be clearly identified’ and were arranged in varying fashions representing a simple nucleolonema. In general, granular components were diffusely disposed in the center of the nucleolus and the fibrillar elements were scattered at the periphery of the nucleolus as knobs (Fig. 3C).
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FIG. 3. Electron micrographs of rat hepatocyte nucleoli after treatment with GalN, FUrd, and Urd. (A) Control. Well-organized nucleolus with sponge-like structure of nucleolonema and evenly distributed granular and tibrillar components (arrows). Nucleolus-associated chromatin (arrowheads) surrounds the nucleolus (x 25,000). (B) FUrd treatment 2 1 and 18 hr before sacrifice. Large bypergranular nucleolus (~24,000). (C) Embedding of liver 6 hr after GalN and 3 hr after Urd injection. Microsegregated nucleolus; knob-like accumulations of the fibpillar components (arrows); granular component is marked (g) (x 19,000). (D) Almost complete reformation of nucleolus 24 hr after GalN with Urd injections at 21 and 18 hr before liver removal (~23,000).
Twenty-four hours after GalN a considerable reformation of nucleolar structure is observed (Fig. 4B; Shinozuka et al., 1973; Enzan et al., 1977). The nucleoli were less compact than normal ones and composed of both granular and fibrillar components, occasionally a rope-like structure of the nucleolonema was observed. Microsegregation was indicated frequently by partial separation of tibrillar and granular components. Administration of FUrd 21 and 18 hr before sacrifice resulted in enlarged, compact, hypergranular nucleoli. They were clearly delimited from other parts of the nucleus by surrounding nucleolus-associated chromatin (Fig. 3B). A similar nucleolar structure was seen when the two doses of FUrd were given after GalN: 24 hr after the amino sugar the nucleoli were enlarged and sharply delimited (Fig. 4D); although the three distinct components were evident,
GALACTOSAMINE
LIVER INJURY AND FLUOROURIDINE
177
FIG. 4. Electron micrograph of hepatocellular nucleoli after treatment with GalN and FUrd. (A) Six hours after GalN injection. Macrosegregated nucleolus composed of dense flbrillar components and a cap (arrowhead) of less dense fibrillar material. Nucleolus-associated chromatin (CH) (~25,000). (B) Twenty-four hours after GalN injection. Microsegregated nucleolus; distinct separation of Ilbrillar (arrow) and granular (g) components (~26,100). (C) Six hours after GalN injection with FUrd treatment at 3 hr. The nucleolonema contains evenly distributed granular and llbrillar material (~25,000). (D) Twenty-four hours after GalN injection with FUrd treatment at 21 and 18 hr before sacrifice. Enlarged (note magnikation) hypergranular nucleolus (x 14,300).
the nucleolonema appeared compact and not prominent. Some cells showed hypergranular nucleoli with a predominance of large granular components. Twenty-four hours after GalN, when UTP deficiency had been reversed by two doses of Urd, the reformation of nucleolar structure was mostly complete (Fig. 3D). Effects of SFluorouridine and Uridine on Galactosamine-Induced Changes in Plasma Lipoproteins The abnormal pattern of plasma lipoproteins observed after GalN administration (Sabesin et al., 1975; Sirowej and Kattermann, 1978) represents an alteration that was prevented or reversed to a large extent by Urd but was intensified by
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FUrd (Fig. 5). The apparent loss of plasma lipoproteins in the electrophoresis pattern 24 hr after GalN with FUrd injected 21 and 18 hr before sacrifice (Fig. 5) was reflected in the reduction of plasma phospholipids and cholesterol to 28 and 21% of the control values, respectively (Table III). FUrd alone, in contrast to Urd, induced a marked reduction in the P-lipoprotein band corresponding to the lowdensity lipoproteins. This alteration was accompanied by a significant fall in the plasma concentrations of phospholipids and cholesterol to 56 and 42% of control, respectively (Table III). The additive effect of GalN and FUrd on phospholipids and cholesterol in plasma was already seen at 12 hr after GalN but it was much more pronounced after 24 hr. In none of the experimental groups (Table III) was there a significant change in the total plasma protein concentration as compared to the control range (55.4 ? 2.1 g/liter). DISCUSSION The pyrimidine nucleoside analog FUrd has in many respects a similar preventive effect on GalN-induced liver injury as Urd and can also be administered as late as 3 hr after the uridylate-trapping amino sugar. The two equimolar doses of both nucleosides administered in a 3-hr interval were required to replenish the uracil nucleotide pools for a sufficient time period (Keppler and Decker, 1971; Table I). As compared to the total FUrd dose used in the chemotherapy of tumor-bearing rats (Heidelberger et al., 1958; Anukarahanonta et al., 1980), this dose of FUrd (two times 3.5 mmole/kg) was more than 26-fold higher. The rats survived the two FUrd doses of 3.5 mmole/kg each for at least 60 hr and the signs of liver toxicity were very limited, in spite of the accumulation of large amounts of 5-fluorouracil nucleotides in this tissue (Table I) and in spite of the key role of liver in the catabolism of FUrd and FUra (Cooper et al., 1972). In agreement with studies in baboons infused with FUra into the hepatic artery (Czerwinski et al., 1975), steatosis was the predominant liver lesion detected by light and electron microscopic analysis and associated with a 3-fold increase in hepatic triglycerides 21 and 18 hr after FUrd treatment (data not shown). Hepatic steatosis is consistent with the decreased concentration of lipoproteins (Fig. 5) as well as phospholipid and cholesterol in plasma (Table III) that are mostly lipoprotein associated. FUrd
a
>
pre-6
>
I3
>
Origin
)
1
r---‘-b'
.7---T
-
FIG. 5. Plasma lipoprotein agarose electrophoresis. Plasma and 18 hr after injection of FUrd or Urd. Sudan black stain.
was obtained
0
24 hr after
GalN,
and 21
GALACTOSAMINE Phospholipids
TABLE III and Total Cholesterol in Plasma 24 hr after Administration 21 and 18 br after SFluorouridine or Uridine Control
Phospholipids Total cholesterol c DXerent
Lomb 49lT91
from the control
152
179
LIVER INJURY AND FLUOROURIDINE
GalN 746 2245 303 267
GdN +
GalN +
FUrd
Urd
mgME(mey+ 105 k 44’
a
of Galactosamine,
FUrd
+ SD; n = 9 to 13) 925 f 244 569 363 f 74
f 146’
208 f 530
Urd
1067 + 174 555 2 89
value by P < 0.001.
and GalN have an additive effect on the increase in hepatic fat and on the loss of some plasma lipoproteins. These two antipyrimidines, however, seem to affect different lipoproteins (Fig. 5) and may interfere with the synthesis and/or glycosylation of different apolipoproteins in liver. GalN has been shown to induce a loss of the C-type apolipoproteins in plasma (Sirowej and Kattermann, 1978). FUrd could interfere with the glycosylation of apolipoproteins if the respective FUDPsugars are inhibitory or inactive substrates. Furthermore, RNA synthesis in the presence of FUTP is capable of producing base-pair transformations (Glazer and Legraverend, 1980) and can result in translational errors (reviewed by Heidelberger, 1975) and could thereby lead to functionally defective apoproteins. In studies on the induction of liver enzyme proteins after administration of S-fluoroorotate, that also leads to an incorporation of FUMP into RNA, it was shown that serine dehydratase is synthesized but without normal enzymatic activity (Mohrenweiser and Pitot, 1974) whereas another enzyme protein, tyrosine aminotransferase, is even increased in activity (Cihak et al., 1973). Our experiments indicate that the reversal by FUrd of GalN-induced UTP and UDP-hexose deficiency restores those processes that are critical for hepatocellular viability during the initial 24 hr after GalN administration. The sequence of events leading to hepatocellular necrosis after a dose of GalN (Keppler, 1976) does therefore not include an interference with the following processes: (a) formation of mature ribosomal RNA (Wilkinson et al., 1971), (b) proper modification of tRNA at the 5-position of uracil (Randerath, 1979), (c) formation of proteins that are synthesized in a defective form after FUMP incorporation into RNA such as serine dehydratase (Mohrenweiser and Pitot, 1974), (d) the thymidylate synthase reaction that is most sensitive to inhibition by FdUMP (Hartmann and Heidelberger, 1961), (e) glycosyltransferase reactions that are affected by FUDP-sugars. FUDP-glucose seems to be an inadequate substitute for UDP-glucose in the glycogen synthase reaction as the hepatic glycogen was resynthesized only poorly ,when FUrd was injected after GalN whereas Urd was capable of restoring the liver glycogen to a large extent (Table I). Studies with isolated sugar transferases are required for a better understanding of the role of FUDP-sugars in glycoprotein and glycolipid synthesis. The GalN-induced block in transcription (Keppler et al., 1974; Konishi et al., 1974; Gajdardjieva et al., 1980) leads to extensive nucleolar fragmentation with a loss of granular components (Shinozuka, 1972; Shinozuka et al., 1473; Enzan et al., 1977; Dimova et al., 1979). This alteration is a common manifestation of ribosomal RNA synthesis inhibition by several agents (Shinozuka, 1972). The effective reversal of this nucleolar lesion by FUrd demonstrates (Fig. 4) that the
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formation of preribosomal RNA, but not its complete maturation, is the prerequisite for the reformation of an intact nucleolar structure. Although maturation of ribosomal RNA and the subsequent formation of new ribosomes remain blocked under this condition, a limited processing of the 45 S preribosomal RNA to 36 and 32 S precursor molecules can proceed (Hadjiolov and Hadjiolova, 1979). FUrd is at least as effective as Urd in the reversal of the GalN-induced nucleolar fragmentation (Figs. 3C and 4C). The hypergranular nucleoli observed 21 and 18 hr after FUrd (Figs. 3B and 4D) may reflect the accumulation of the ribosomal RNA precursors that cannot be processed further and released into the cytosol. ACKNOWLEDGMENTS We are indebted to Christa Holstege for her excellent technical assistance and to Karl Happersberger for his expert help in all animal experiments.
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