Aging decreases rat liver regeneration. We (1) compared the expression of ornithine decarboxylase (ODC), a critical enzyme for liver regeneration, and polyamine levels in regenerating liver of 6-week-old and 1-year-old rats and (2) evaluated the effect of exogenous putrescine supplementation on liver regeneration in 1-year-old rats. ODC messenger ribonucleic acid (mRNA) transcript sizes were the same in rats of both ages. ODC mRNA content and enzyme activity were higher in the younger rats; however, magnitudes of increase after partial hepatectomy were greater in the older rats. From peak levels, the rate of decline of the mRNA was slower in the older rats, but enzyme activity declined at the same rate in both ages. ODC apoenzyme content was significantly less in normal liver tissue from 1-year-old rats, but there was little change after partial hepatectomy in rats of either age. No change in ODC transcriptional activity was found. Hepatic putrescine levels were lower in 48 hours regenerating liver tissue from 1-year-old rats. To determine whether supplemental putrescine would increase liver regeneration in 1-year-old rats, putrescine (600 mumol/kg IP every 4 hours) was administered beginning 4 days before or at the time of partial hepatectomy. This raised polyamine levels and decreased ODC activity significantly, but there was no change in regenerating liver weight, total DNA and RNA content, and tritiated thymidine incorporation at 48 hours. These results indicate that ODC expression is different and polyamine levels are lower in 1-year-old rats than in 6-week-old rats. However, putrescine supplementation that is sufficient to decrease ODC activity has no apparent effect on regeneration.

Polyamines are considered critical for cell proliferation. During liver regeneration in the rat, ornithine decarboxylase (ODC) mRNA and enzyme activity and polyamines (primarily putrescine and spermidine) are known to increase substantially. We examined the effect of inhibition of polyamine synthesis with alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of the ODC enzyme, on regenerating liver weight and total DNA, RNA, and protein, [3H]thymidine and [14C]leucine incorporation, number of mitotic figures, and putrescine, spermidine, and spermine contents. Rats received DFMO beginning 4 days before or immediately after two-thirds partial hepatectomy. In control rats, ODC activity, putrescine, and spermidine increased significantly during regeneration, whereas spermine was unchanged. In rats receiving DFMO, ODC and putrescine changed minimally but spermidine increased as usual. Spermine levels were modestly higher in rats receiving DFMO beginning 4 days before partial hepatectomy. However, despite ODC inhibition and substantially lower levels of putrescine, the course of liver regeneration in rats treated with DFMO was not affected. Total liver mass, DNA, RNA, and protein increased over 5 days equally in rats receiving DFMO and control rats. In addition, there were no differences in [3H]thymidine incorporation into DNA, [14C]leucine incorporation into protein or mitotic indexes between DFMO-treated and control rats at 24 and 48 h after partial hepatectomy. These results suggest that the well-known increases in ODC activity and polyamines that occur during regeneration are not required for liver to undergo its proliferative response to partial hepatectomy.

We studied the effect of aging on rat liver regeneration. We compared the time course of total hepatic mass, DNA and RNA accumulation, and thymidine kinase (TK) messenger RNA (mRNA) content and enzyme activity, after two-thirds partial hepatectomy in 6-week-old (young adult) and 1-year-old rats. Whereas 6-week-old rats had completely regenerated all liver mass, DNA, and RNA by 7 days, the regenerating 1-year-old rat livers at 7 days contained only 60% to 70% of the mass, DNA, and RNA in the normal 1-year-old rat liver. However, rates of tissue regeneration during this time were very similar in both ages. At 28 days the weight and RNA content of the 1-year-old rat livers were 93% of normal, but total DNA was still reduced, at 78% of normal. In the younger rats TK mRNA content and enzyme activity increased substantially after partial hepatectomy and were observed to peak at 24 hours. In the 1-year-old rats TK mRNA was less abundant in normal liver and the peak level observed was lower and delayed until 48 hours. Nonetheless, the incremental increase from baseline to peak was greater in the 1-year-old than in the 6-week-old rats. In contrast to the mRNA, TK activity peaked at 24 hours, but at substantially lower levels than in the 6-week-old rats. Rates of disappearance of TK mRNA and enzyme activity after peak levels were not significantly different by age.(ABSTRACT TRUNCATED AT 250 WORDS)

Ornithine decarboxylase and thymidine kinase are enzymes that increase in activity in regenerating liver. We found that both activities and mRNA levels for these enzymes increase significantly after 70% partial hepatectomy in the rat. After sham hepatectomy (laparotomy) there were significant decreases in activity; however, mRNA content was unaltered. Similar decreases in enzyme activity, without changes in mRNA content, were found with pair-feeding, and additional decreases in activity after starvation. In contrast to previous reports of no change in ornithine decarboxylase and thymidine kinase after sham hepatectomy, the present results indicate that decreases occur. This may be mediated by the decrease in food intake after surgery. Dietary factors may be important in the physiologic regulation of these enzymes in the liver.

The sequential and comparative ultrastructural features of regenerating rat liver following onelobe, two-lobe, and subtotal hepatectomy were studied. All three groups demonstrated glycogen depletion at the 12 h post-hepatectomy interval with reaccumulation occurring at 24 h in the first two groups but not until 72 h in the subtotal hepatectomy group. Other hepatocellular alterations attributed to regenerative activity were similar in the three groups, however the onset and magnitude of those changes occurring in the two-lobe and subtotally hepatectomized rats differed significantly from those alterations occurring in the one-lobe hepatectomy group. These changes were characterized by a greater and more prolonged mitotic activity, increased proliferation of RER and smooth endoplasmic reticulum (SER), and by increased Golgi bodies. These two groups also manifested greater hepatocellular accumulation of phagolysosomes, myelin figures, and lipid bodies when compared with the one-lobe hepatectomy group.

Hepatic thymidine kinase (TK) and ornithine decarboxylase (ODC) activities were used to quantify the regenerative response to injury with galactosamine. After massive damage with 1000 mg/kg galactosamine, TK activity (DNA synthesis) peaked between 62 and 120 hours. This peak of activity was depressed as much as 91% by six subcoma doses of dimethyl disulfide (DMDS) given between 24 hours and 64 hours after galactosamine administration. Similar doses of octanoic acid (OA) had no effect, and doses of NH4Cl had no effect except at 120 hours. The first peak of ODC activity (initiation of cell growth) at 45 hours was depressed about 60% by six subcoma doses of NH4Cl or DMDS injected between 27 hours and 42 hours. OA again had no effect. After 400 mg/kg galactosamine, a narrow but high peak of TK activity occurred at 62 hours. This peak of activity was depressed more than 50% by six subcoma doses of NH4Cl, OA, or DMDS given between 24 hours and 54 hours. The first peak of ODC activity at 36 hours was similarly reduced by more than 50% by similar doses of each of the toxins given between 24 hours and 34 hours. The overt neurologic effects of the toxins were dissipated within 1 hour of each injection. The depressive effect of NH4Cl and OA on TK and ODC activities during regeneration after massive centrolobular injury with acetaminophen was more consistently present and more extensive than that seen after injury with galactosamine.

Persistent slight or modest increments in blood ammonia level resulting from continuous intravenous infusion of NH4+ depressed hepatic thymidine kinase (TK) activity (DNA synthesis) by 30% after two-lobe (70%) hepatectomy, by 32% after subtotal (90%) hepatectomy, by 80% after massive injury with 1400 mg/kg acetaminophen, and by 92% after massive injury with 1000 mg/kg galactosamine. Ornithine decarboxylase activity (reflecting initiation of regeneration) was similarly depressed by 65% after 70% hepatectomy, by 58% after acetaminophen, and by 87% after galactosamine. It was not depressed after 90% hepatectomy. Intermittent marked but transient increments in blood ammonia level resulted in similar but slightly smaller enzyme reductions after the injuries with the toxins, and a reduction of 92% in TK activity after 70% hepatectomy. Thus, after toxic injury, both regenerative enzymes were substantially depressed by excess ammonia, whether present transiently in large amounts or persistently in small amounts. After partial hepatectomy, TK activity was similarly depressed by the large transient amounts of ammonia but was less affected by the persistent smaller amounts.

Hepatic regenerative enzyme (thymidine kinase and ornithine decarboxylase) activities were significantly depressed by subcoma doses of the hepatic failure toxins (NH4+, octanoic acid, and dimethyl disulfide) after selective injury with allyl alcohol. The inhibitory effect of NH4+ was greater than that of dimethyl disulfide, even though the neurologic effects of dimethyl disulfide were approximately comparable with those of the NH4+. There appeared to be a delay in the full expression of the depressive effects of octanoic acid and dimethyl disulfide. The resistance to these two toxins, particularly dimethyl disulfide, may reflect the resistance to injury of the oxidative processes prominent in periportal hepatocyte mitochondria. In comparison with pericentral injury or two-lobe hepatectomy, periportal injury seemed equally susceptible to regenerative enzyme inhibition by NH4+ but less susceptible to the effect of octanoic acid and dimethyl disulfide.

Massive liver injury with 1000 mg/kg D-galactosamine was followed by a broad peak of thymidine kinase (TK) activity over 62 to 120 hours. The highest mean value was a 29-fold increase in activity. Three peaks of ornithine decarboxylase (ODC) activity were observed over the same time span. The first peak occurred at 45 hours, showing a sevenfold to eightfold increase in activity. Histologic evidence of necrosis peaked at 12 to 24 hours and was prominent over 54 hours. Periportal inflammation in response to the cellular injury was prominent from 12 to 96 hours and peaked at 45 to 62 hours. The curve of mitoses peaked at the same time as that of TK activity but was only 68% as extensive. The first peak of ODC activity occurred before there was any significant presence of mitoses or of TK activity. A smaller dose of 400 mg/kg resulted in a narrow peak of TK activity at 62 hours that was a 37-fold increase in activity. The total TK response was 43% of that seen after the larger dose, about in proportion to the dose given. The first ODC peak, representing the earliest evidence of regeneration, was earlier and more prominent after the 400 mg/kg dose. On the basis of areas under the curves, the amounts of necrosis and of periportal inflammation after 400 mg/kg were 36% of those seen after 1000 mg/kg. Corresponding figures for other response curves were 29% for mitoses, 33% for serum SGPT, and 71% for total ODC activity.(ABSTRACT TRUNCATED AT 250 WORDS)

The neuromodulators, adenosine, serotonin, and glycine, did not alter the course of hepatic encephalopathy (HE) that followed a portacaval shunt and hepatic artery ligation in rats. The substances were instilled into the brain ventricle through an intraventricular cannula in doses that affect other aspects of behavior in the normal rat (adenosine, suppression of food intake; serotonin, loss of muscle strength and ataxia; glycine, leaning and circling). A subconvulsive dose of the glycine antagonist, strychnine, also had no effect on the course of HE. A large dose of the adenosine antagonist, caffeine, had a depressive rather than excitatory effect and shortened the time taken to induction of coma. These studies and a similar previous one with γ-aminobutyric acid (GABA) suggest that the inhibitory neuromodulators do not have a prominent role in the pathogenesis of hepatic coma.

The specific binding of γ-aminobutyric acid (GABA) to synaptic membranes prepared from the brains of rats with acute liver failure due to portacaval shunt and hepatic artery ligation was measured. No changes in the affinity or the density of the low- and high-affinity binding sites of the GABA receptor were observed, indicating that this model of acute hepatic encephalopathy is apparently not associated with altered GABAergic neurotransmission.

The progressive course of hepatic encephalopathy developing in rats after massive hepatic ischemia due to hepatic artery ligation within 48 hr of a portacaval shunt was not altered by the injection of a benzodiazepine antagonist, CGS 8216, in a dose that was sufficient to reverse diazepam-induced coma quickly. The onset of hepatic coma was shortened 20 to 25% by the antagonist, rather than being delayed, as would be expected if hepatic coma were due to a γ-aminobutyric acid (GABA)-ergic effect. The neural binding of GABA by brains from rats in deep hepatic coma was unaffected by the injection of the benzodiazepine antagonist.

Pericentral and periportal liver injuries involving less than 50% of the parenchyma were produced with acetaminophen and allyl alcohol, respectively. Doses were selected to produce comparable peak serum malate dehydrogenase, sorbitol dehydrogenase, and SGPT activities. The regenerative response was assessed by serial measurements of hepatic thymidine kinase (TK) activity and ornithine decarboxylase (ODC) activity. The initial responses reflected in ODC activity were more or less similar. However, the ultimate regenerative response reflected by TK activity was almost three times as great after periportal injury as after pericentral injury, after allowing for differences in the extent of necrosis. Histologic examination also showed greater mitotic and tissue reparative responses after periportal injury. These results suggest that the concept of hepatocellular heterogeneity applies to the regenerative response of liver cells as well as the metabolic functions previously identified.

Four injections of subcoma doses of ammonium acetate, octanoic acid or dimethyl disulfide during the first 24 hr after two-lobe hepatectomy in normal rats markedly depressed DNA synthesis as reflected by liver thymidine kinase activity or the incorporation of tritiated thymidine into hepatic DNA. Recovery from the depressant effects of the three toxins took 16 to 28 hr. Similar doses of the same toxins injected hourly for 3 or 5 hr after the two-lobe hepatectomy had similar depressant effects on the early peak of ornithine decarboxylase activity measured at 4 or 6 hr. Recovery occurred within 3 hr perhaps because of the very short half-life of ornithine decarboxylase and its rapid regeneration time. These observations may have implications for the lack of regeneration observed in many patients with fulminant hepatic failure who have accumulated sufficient ammonia, methanethiol and fatty acids over periods of days or weeks to become encephalopathic.

Massive liver injury was produced in fasting male Sprague-Dawley rats weighing 200 +/- 25 gm each by gastric administration of 1400 mg/kg acetaminophen. The time sequence of changes in liver ornithine decarboxylase (ODC) activity, which reflects the earliest phases of cell multiplication, liver thymidine kinase (TK) activity, which reflects DNA synthesis, and liver histology (necrosis, mitosis, and repair processes) was recorded. ODC showed the usual biphasic response. By 12 hours, it reached its first peak, a six- to eightfold increase. At this time there was no histologic evidence of necrosis, and serum malate dehydrogenase (MDH), sorbitol dehydrogenase (SDH), and alanine aminotransferase (SGPT) were normal. During the next 12 hours ODC decreased by 60% to 70% and cellular necrosis became evident, and reached a peak at 24 to 36 hours, as did serum MDH, SDH, and SGPT. The serum enzymes fell precipitously at 48 hours, but the histologic evidence of necrosis subsided gradually over 60 hours. The secondary ODC peak, a fourfold increase, coincided with rising activity of TK, which increased 25- to 35-fold over 54 to 72 hours, and then subsided. At 54 hours, when DNA synthesis had already peaked, there was no histologic evidence of repair other than mitoses. However, within the next 6 hours, evidences of repair became prominent, and remained so for another 36 hours before subsiding. Thus, with acetaminophen injury, the initial phases in preparation for cell multiplication occurred before histologic evidence of injury was apparent, and DNA synthesis peaked before other evidence of tissue repair became evident.(ABSTRACT TRUNCATED AT 250 WORDS)