Pro-oxidant and antioxidant mechanisms of etoposide in HL-60 cells: role of myeloperoxidase.

Etoposide is an effective anticancer agent whose antitumor activity is associated with its phenolic E-ring, which can participate in intracellular redox cycling reactions. Myeloperoxidase (MPO)-catalyzed one-electron oxidation of the etoposide phenolic ring and/or interaction of this phenolic moiety with reactive radicals yields its phenoxyl radical, whose reactivity may determine the pro- or antioxidant effects of this molecule in cells. Using MPO-rich HL-60 cells, we directly demonstrated that both anti- and pro-oxidant activities of etoposide are realized in cells. Etoposide acted as an effective radical scavenger and antioxidant protector of phosphatidylethanolamine, phosphatidylcholine, and other intracellular phospholipids against H2O2-induced oxidation in HL-60 cells with constitutively high MPO activity and in HL-60 cells depleted of MPO by an inhibitor of heme synthesis, succinyl acetone. MPO-catalyzed production of etoposide phenoxyl radicals observed directly in HL-60 cells by electron paramagnetic resonance (EPR) did not result in oxidation of these membrane phospholipids, suggesting that the radicals were not reactive enough to trigger lipid oxidation. MPO-dependent pro-oxidant activity of etoposide was directly demonstrated by (a) the ability of intracellular reduced glutathione (GSH) to eliminate EPR-detectable etoposide phenoxyl radicals, (b) the ability of etoposide phenoxyl radicals to oxidize GSH and protein thiols (after preliminary depletion of intracellular GSH with a maleimide reagent, ThioGlo-1), and (c) the disappearance of these effects after depletion of MPO by pretreatment of cells with succinyl acetone. In addition, titration of intracellular GSH (in intact cells) using the maleimide reagent ThioGlo-1 resulted in remarkably augmented EPR-detectable etoposide phenoxyl radicals and enhanced etoposide-induced topoisomerase II-DNA covalent complexes. In conclusion, the phenolic moiety of etoposide acts as an effective free radical scavenger, accounting for its antioxidant action. Whereas one-electron oxidation of etoposide by free radical scavenging and/or by MPO results in a phenoxyl radical with low reactivity toward lipids, its high reactivity toward thiols is a determinant of its pro-oxidant effects in HL-60 cells.

Therapeutic effect of S-allylmercaptocysteine on acetaminophen-induced liver injury in mice.

S-allylmercaptocysteine is one of the water-soluble organosulfur compounds in ethanol extracts of garlic (Allium sativum L.). We had demonstrated earlier that treatment with S-allylmercaptocysteine before acetaminophen administration protects mice against acetaminophen-induced hepatotoxicity. In this study, we examined the therapeutic effect of S-allylmercaptocysteine treatment after acetaminophen administration. A single dose of S-allylmercaptocysteine (200 mg/kg, p.o.) to mice 0.5 h after acetaminophen administration (500 mg/kg, p.o.) significantly suppressed both the increase in plasma alanine aminotransferase activity and the hepatic necrosis, and also reduced acetaminophen-induced mortality from 43% to 0%. These data indicate that S-allylmercaptocysteine is useful as an antidote for acetaminophen overdose. S-allylmercaptocysteine significantly suppressed hepatic cytochrome P450 2E1 (CYP2E1) activity and induction of inducible 70-kDa heat shock protein, a marker of acetaminophen arylation of protein. These results suggest that S-allylmercaptocysteine exerts its protective effect by inhibition of CYP2E1 activity, which leads to the suppression of acetaminophen arylation of hepatic protein.

Protection by polaprezinc, an anti-ulcer drug, against indomethacin-induced apoptosis in rat gastric mucosal cells.

Polaprezinc [N-(3-aminopropionyl)-L-histidinato zinc] (PZ), an anti-ulcer drug, is a chelate compound consisting of zinc and L-carnosine. PZ has been shown to prevent gastric mucosal injury. In the present study, we investigated the inhibitory effect of PZ on indomethacin (IND)-induced apoptosis in a rat gastric mucosal cell line, RGM1. Pretreatment with PZ suppressed caspase-3 activation and subsequent apoptosis in the cells exposed to 500 microM IND in a dose-dependent manner, and 50 microM PZ exhibited the maximum inhibitory effect. Among PZ subcomponents, zinc but not L-carnosine played a pivotal role in this antiapoptotic function. PZ did not affect mitochondrial cytochrome c release upstream of caspase-3 activation in the IND-induced apoptotic signal pathway. Treatment with 500 microM IND evidently produced reactive oxygen species (ROS) in RGM1 cells. However, PZ did not scavenge ROS in IND-treated cells. Moreover, N-acetylL-cysteine, a potent antioxidant, inhibited ROS generation but did not suppress apoptosis in RGM1 cells exposed to IND. These observations demonstrate a novel pharmacological action of PZ; i.e., that PZ, and in particular its zinc subcomponent, inhibits apoptosis via inhibition of caspase-3 activation but not antioxidant activity.

Mitochondrial cytochrome c release and caspase-3-like protease activation during indomethacin-induced apoptosis in rat gastric mucosal cells.

Indomethacin (IND), a nonsteroidal anti-inflammatory drug, has been known to cause gastric mucosal injury as a side effect. Using a rat gastric mucosal cell line, RGM1, we determined whether apoptosis is involved in IND-mediated gastropathy, and whether caspase activation and mitochondrial cytochrome c release play an important role in producing apoptosis of IND-treated RGM1 cells in the presence of serum. IND caused caspase-3-like protease activation followed by apoptosis in a dose- and time-dependent manner. Caspase-1-like protease activity did not change during IND-induced apoptosis. IND also increased mitochondrial cytochrome c release in a time-dependent fashion. Mitochondrial cytochrome c efflux occurred just before or at the same time as caspase-3-like protease activation, and preceded the increase in apoptotic cell numbers. Z-VAD-FMK, a caspase inhibitor, inhibited both the increase in caspase-3-like protease activity and apoptosis in IND-treated RGM1 cells but did not affect caspase-1-like protease activity or mitochondrial cytochrome c release. These observations suggest that the apoptosis of gastric mucosal cells could be involved in IND-induced gastropathy, that cytochrome c is released from mitochondria into the cytosol during the early phase of IND-mediated apoptosis, and that subsequent activation of caspase-3-like protease, but not caspase-1-like protease, is required for the execution of apoptosis.

Quinolizin-coumarins as physical enhancers of chemiluminescence during lipid peroxidation in live HL-60 cells.

We investigated whether physical enhancers of low-level chemiluminescence-coumarin laser dyes C-314, C-334, and C-525--may be used to monitor interactions of lipid peroxyl radicals during lipid peroxidation in live cells. We present data demonstrating that two quinolizin-substituted coumarins--C-525 and C-334--can be integrated into HL-60 cells and successfully used as physical enhancers of chemiluminescence induced by the lipid soluble azo-initiator 2,2'-azobis(2,4-dimethyl-valeronitrile) (AMVN). Coumarins did not inhibit AMVN-induced peroxidation of membrane phospholipids in HL-60 cells, and no consumption of these coumarins occurred in the course of AMVN-induced oxidative stress. Redox status, evaluated by intracellular GSH content, remained unchanged after treatment with the coumarins. tert-Butyl hydroperoxide and cumene hydroperoxide (more hydrophilic oxidants) induced a lower chemiluminescence signal with both coumarins. Viability of HL-60 cells was not affected by coumarins both in the presence and in the absence of oxidants. Based on these results we conclude that quinolizin-substituted coumarins represent a promising class of physical enhancers of chemiluminescence for monitoring free radical peroxidation in live cells.

Hydrogen peroxide-induced apoptosis in HL-60 cells requires caspase-3 activation.

Apoptosis has been associated with oxidative stress in biological systems. Caspases have been considered to play a pivotal role in the execution phase of apoptosis. However, which caspases function as executioners in reactive oxygen species (ROS)-induced apoptosis is not known. The present study was performed to identify the major caspases acting in ROS-induced apoptosis. Treatment of HL-60 cells with 50 microM hydrogen peroxide (H2O2) for 4 h induced the morphological changes such as condensed and/or fragmented nuclei, increase in caspase-3 subfamily protease activities, reduction of the procaspase-3 and a DNA fragmentation. To determine the role of caspases in H2O2-induced apoptosis, caspase inhibitors, acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone (Ac-YVAD-cmk), acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) and acetyl-Val-Glu-Ile-Asp-aldehyde (Ac-VEID-CHO), selective for caspase-1 subfamily, caspase-3 subfamily and caspase-6, respectively, were loaded into the cells using an osmotic lysis of pinosomes method. Of these caspase inhibitors, only Ac-DEVD-CHO completely blocked morphological changes, caspase-3 subfamily protease activation and DNA ladder formation in H2O2-treated HL-60 cells. This inhibitory effect was dose-dependent. These results suggest that caspase-3, but not caspase-1 is required for commitment to ROS-triggered apoptosis.

Mechanisms of protection by S-allylmercaptocysteine against acetaminophen-induced liver injury in mice.

S-Allylmercaptocysteine (SAMC), one of the water-soluble organosulfur compounds in ethanol extracts of garlic (Allium sativum L.), has been shown to protect mice against acetaminophen (APAP)-induced liver injury. In this study, we examined the mechanisms underlying this hepatoprotection. SAMC (100 mg/kg, p.o.) given 2 and 24 hr before APAP administration (500 mg/kg, p.o.) suppressed the plasma alanine aminotransferase activity increases 3 to 12 hr after APAP administration significantly. The hepatic reduced glutathione levels of vehicle-pretreated mice decreased 1 to 6 hr after APAP administration, but SAMC pretreatment suppressed the reductions 1 to 6 hr after APAP administration significantly. These inhibitory effects of SAMC were dose-dependent (50-200 mg/kg) 6 hr after APAP administration. As SAMC pretreatment (50-200 mg/kg) suppressed hepatic cytochrome P450 2E1-dependent N-nitrosodimethylamine demethylase activity significantly in a dose-dependent manner, we suggest that one of its protective mechanisms is inhibition of cytochrome P450 2E1 activity. SAMC pretreatment also suppressed the increase in hepatic lipid peroxidation and the decrease in hepatic reduced coenzyme Q9 (CoQ9H2) levels 6 hr after APAP administration. The hepatic CoQ9H2 content of the SAMC pretreatment group was maintained at the normal level. Therefore, we suggest that another hepatoprotective mechanism of SAMC may be attributable to its antioxidant activity.

Protective effect of UW solution on postischemic injury in rat liver: suppression of reduction in hepatic antioxidants during reperfusion.

Preservation with University of Wisconsin (UW) solution can maintain liver graft function and produces survival rates of recipients higher than that with Euro Collins (EC) solution. To explore the underlying mechanisms, we transplanted rat livers following cold preservation with EC or UW solution for 18 hr, and measured hepatic adenine nucleotide levels, the percentage of water content, lactate levels, and endogenous antioxidant levels (alpha-tocopherol [alpha-Toc], reduced coenzyme Q9 [CoQ9H2], reduced coenzyme Q10, [CoQ1OH2] and reduced glutathione [GSH] during preservation and after transplantation. The adenosine triphosphate levels of the liver grafts preserved with UW solution recovered after reperfusion more rapidly and reached a higher level than those preserved with EC solution. UW solution caused a reduction in hepatic water content during preservation. Conversely, EC solution induced remarkable tissue edema. In addition, UW solution reduced the rate of hepatic lactate production both during preservation and after reperfusion. The concentrations of hepatic GSH, alpha-Toc, CoQ9H2, and CoQ1OH2 immediately after the graftectomy, and after the 18 hr of preservation with both EC and UW solutions, did not differ from those in the normal liver, and decreased only after transplantation. However, UW solution suppressed significantly the reduction in hepatic GSH, alpha-Toc, and CoQ9H2 after reperfusion, compared with EC solution. These results suggest that long-term cold storage induces tissue edema, reflecting a disturbance of the microcirculation during preservation, followed by parenchymal cell damage mediated by free radicals after reperfusion. The protective effects of UW solution could be attributable to the inhibition of free radical production after reperfusion.

alpha-Tocopherol pretreatment protects the endocrine function of grafts against ischemic damage during heterotopic pancreatic transplantation.

Post-ischemic injury is one of the most important problems affecting successful organ procurement and transplantation. The present study was performed to determine whether alpha-tocopherol can protect the endocrine function of pancreatic grafts against ischemia-reperfusion injury during rat heterotopic pancreatic transplantation. Rats with streptozotocin-induced hyperglycemia were used as recipients. The donor pancreas was removed and subjected to warm ischemia at 37 degrees C for 30, 60, 90 and 120 min, and then transplanted into a recipient. A 30-min period of warm ischemia did not impair the endocrine function of the pancreatic grafts, which was assessed by measuring the blood glucose levels and glucose decay constants (K), and a 60-min period of warm ischemia was considered to be the critical period for reversible tissue damage. Pretreatment with alpha-tocopherol (20 mg/kg/day, i.v.) for seven days before graftectomy significantly decreased blood glucose levels to less than 200 mg/dl and significantly increased K values in the recipient rats after transplantation when compared with placebo pretreatment. These results suggest that alpha-tocopherol pretreatment can protect the endocrine function of pancreatic grafts against injury due to warm ischemia followed by reperfusion.

Acetaminophen-induced hepatic injury in mice: the role of lipid peroxidation and effects of pretreatment with coenzyme Q10 and alpha-tocopherol.

This study was performed to determine whether oxidative stress contributed to the initiation or progression of hepatic injury produced by acetaminophen (APAP). Treatment of fasted mice with APAP (400 mg/kg, I.P.) led to hepatic injury as indicated by a marked elevation of plasma alanine aminotransferase (ALT). APAP caused an increased amount of thiobarbituric acid-reactive substance (TBARS), which was accompanied by a loss of reduced forms of coenzyme Q9 (CoQ9H2) and coenzyme Q10 (CoQ10H2) functioning as antioxidants. APAP also markedly decreased hepatic reduced glutathione (GSH) levels. Pretreatment with CoQ10 (5 mg/kg, I.V.) reduced hepatic TBARS levels to 30% and plasma ALT levels to 26% of placebo pretreatment levels without affecting hepatic GSH levels at 3 h of APAP treatment. alpha-Tocopherol (alpha-Toc) (20 mg/kg, I.V.) pretreatment also reduced hepatic TBARS levels to 13% and plasma ALT levels to 27% of placebo pretreatment levels without affecting hepatic GSH levels. These results suggest that oxidative stress followed by lipid peroxidation might play a role in the pathogenesis of APAP-induced hepatic injury, and pretreatment with lipid-soluble antioxidants such as CoQ10 and alpha-Toc can limit hepatic injury produced by APAP.

A case of gas-containing liver abscess associated with emphysematous change in the gallbladder.

We describe a 77-year-old man with diabetes mellitus who developed a large gas-containing pyogenic liver abscess after admission. Mild elevation of serum biliary enzyme levels suggested probable biliary trouble on admission. Ultrasonography and computed tomography showed a large abscess of the liver with gas formation and the presence of gas within the lumina of the gallbladder and biliary tract when the patient had fever, leukocytosis and evidence of hepato-renal dysfunction. These findings suggest that the large liver abscess may have developed as a result of emphysematous cholecystitis.

Protective effects of coenzyme Q10 and α-tocopherol against free radical-mediated liver cell injury.

In an attempt to provide further confirmation of the antioxidant role of reduced form of coenzyme Q homologue (CoQnH2) and α-tocopherol (α-Toc), we incubated isolated rat hepatocytes with a water-soluble radical initiator, 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH) in the presence or absence of exogenously added coenzyme Q10 (CoQ10) or α-Toc for 3 h at 37°C under an atmosphere of 95% oxygen and 5% carbon dioxide. In the control experiment without adding AAPH it was confirmed that added CoQ10 and α-Toc were incorporated into the cells and some CoQ10 were converted to CoQ10H2. Incubation of hepatocytes with 50 mM AAPH resulted in the formation of thiobarbituric acid-reactive substances and the decrease in cell viability and both were inhibited by exogenously added CoQ10 or α-Toc in a dose-dependent manner. The decrease in endogenous CoQ9H2 and α-Toc levels was observed by the addition of AAPH. Addition of CoQ10 inhibited the oxidation of CoQ9H2 to CoQ9 dose-dependently while the addition of α-Toc did not. These data suggest that both CoQnH2 and α-Toc act as antioxidants and can inhibit free radical-mediated cell injury.

Iron ion induces mitochondrial DNA damage in HTC rat hepatoma cell culture--role of antioxidants in mitochondrial DNA protection from oxidative stresses.

The mitochondrial DNA (mtDNA) is exposed to the reactive oxygen species generated in the mitochondrial electron transfer system. While mitochondrial superoxide dismutase (Mn-SOD) scavenges superoxide anions to prevent damage of mtDNA, excess amount of reactive oxygen generated by quinone compounds impairs the mtDNA, in which involvement of iron ion-catalyzed reactions is suggested. In this communication, we report that the mtDNA in HTC rat hepatoma cells was markedly damaged in the presence of either ferrous (Fe2+) or ferric (Fe3+) iron in the culture medium. The mtDNA of HTC cells was damaged in the presence of 100 microM of iron ions in the culture medium within 3 h. There was no significant difference in the potency of Fe2+ and Fe3+. Deferoxamine, a Fe(3+)-specific chelating reagent, blocked the iron ion-induced mtDNA damage completely. The mtDNA in rat hepatocyte primary culture was, on the other hand, not damaged by either Fe2+ or Fe3+. To understand the difference between iron ion-induced damage of mtDNA in HTC cells and in the primary hepatocytes, the Mn-SOD activity and lipid-soluble antioxidant contents were compared. The Mn-SOD activity in HTC cells was markedly lower (< 0.02 U/mg cell protein) than that in rat hepatocyte primary cell cultures (1.5 U/mg cell protein). Immunoreactive Mn-SOD content in HTC cells was also lower (21 ng/mg cell protein) than in the primary cultures (99 ng/mg cell protein). HTC cells contain much smaller amounts (3-11% of that of normal rat hepatocytes) of alpha-tocopherol and coenzyme Q homologs (CoQn). These results suggest that reactive oxygen species produced by iron ion impaired mtDNA of HTC cells, in which antioxidant activity was markedly decreased.

Antioxidant role of cellular reduced coenzyme Q homologs and alpha-tocopherol in free radical-induced injury of hepatocytes isolated from rats fed diets with different vitamin E contents.

Reduced coenzyme Q9 (CoQ9H2) and reduced coenzyme Q10 (CoQ10H2) as well as alpha-tocopherol (alpha-Toc) are known to be potent lipid-soluble antioxidants in mammalian tissues. Reduced coenzyme Q homolog (CoQnH2) appears to show antioxidant activity independent of that of alpha-Toc (Matsura, T., Yamada, K. and Kawasaki, T. (1992) Biochim. Biophys. Acta 1123, 309-315). To further confirm this, we have studied the antioxidant role of cellular CoQnH2 and alpha-Toc using hepatocytes isolated from rats fed diets containing deficient, sufficient, and excess amounts of vitamin E (VE). Cellular damage was induced with a hydrophilic radical initiator, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). The concentration of alpha-Toc in VE-deficient hepatocytes was approximately 1/12 that in VE-sufficient hepatocytes, whereas the concentration of alpha-Toc in VE-excess hepatocytes was approximately 7-fold that in VE-sufficient hepatocytes. The molar ratios of alpha-Toc to CoQnH2 (CoQ9H2 plus CoQ10H2) in VE-deficient, sufficient and excess cells were 0.03, 0.33 and 2, respectively. In the hepatocytes in these three dietary groups, alpha-Toc status had little effect on the concentration of CoQ homologs. These hepatocytes were incubated with 50 mM AAPH for 4 h. The cell viability in all groups of hepatocytes decreased rapidly after 3 h of AAPH treatment, and was associated with the increase of lipid peroxides. The loss of cell viability and the increase of lipid peroxidation in VE-deficient cells were more pronounced than those in the hepatocytes of the other two groups. The endogenous CoQ9H2 content of each group of hepatocytes decreased linearly with a reciprocal increase in oxidized CoQ9 after addition of AAPH, whereas the decrease of endogenous CoQ10H2 in each group during AAPH treatment was much less than that of endogenous CoQ9H2. alpha-Toc in the three VE dietary groups of hepatocytes was also consumed without a time lag after addition of AAPH, and it was not spared by CoQnH2, even in VE-deficient cells where the CoQnH2 concentration was 38-fold that of alpha-Toc. These results indicate that CoQnH2, especially. CoQ9H2, is a lipid-soluble antioxidant, which is as effective as alpha-Toc in rat hepatocytes under the conditions employed in this study, and acts independently of alpha-Toc to inhibit lipid peroxidation.

Difference in antioxidant activity between reduced coenzyme Q9 and reduced coenzyme Q10 in the cell: studies with isolated rat and guinea pig hepatocytes treated with a water-soluble radical initiator.

A possible difference in antioxidant activity between reduced coenzyme Q9 (CoQ9H2) and reduced coenzyme Q10 (CoQ10H2) in animal cells was studied by incubation of hepatocytes with a hydrophilic radical initiator, 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH). Two kinds of hepatocytes differing in their content of CoQ homologs were used: rat, total (oxidized plus reduced) CoQ9: total CoQ10 6:1, guinea pig, 1:5. The sum of total CoQ9 and CoQ10 in rat and guinea-pig hepatocytes was about 780 and 400 pmol/mg protein, respectively. The concentration of CoQ9H2 in rat hepatocytes decreased linearly after the addition of AAPH, whereas that of oxidized CoQ9 showed a reciprocal increase. No loss of cell viability or increase of lipid peroxidation was observed until most of the CoQ9H2 had been consumed. Cellular CoQ9H2 was consumed probably through scavenging of lipid peroxyl radicals produced by incubation with AAPH. On the other hand, CoQ10H2 was not significantly consumed in the AAPH-treated rat hepatocytes during incubation compared with the control cells. In guinea-pig hepatocytes, cellular CoQ10H2 as well as CoQ9H2 was consumed by addition of AAPH. alpha-Tocopherol also showed linear consumption with incubation time regardless of the cell types used. It is concluded that CoQ9H2, together with alpha-tocopherol, constantly acts as a potential antioxidant in hepatocytes when incubated with AAPH, whereas CoQ10H2 mainly exhibits its antioxidant activity in cells containing CoQ10 as the predominant CoQ homolog.

Changes in the content and intracellular distribution of coenzyme Q homologs in rabbit liver during growth.

In order to determine whether coenzyme Q (CoQ) homologs which coexist in mammals play the same or different roles, the concentrations of coenzyme Q9 (CoQ9) and coenzyme Q10 (CoQ10) were analyzed in Japanese White (JW) rabbit tissues during growth, together with the intracellular distribution of these two CoQ homologs. In liver %CoQ9 (total [CoQ9] X 100/total [CoQ9] + total [CoQ10]) was approx. 40% until 3 weeks after birth, and then gradually decreased to 20%. In kidney, %CoQ9 decreased from 8% (1 week) to 1% (7 weeks). In heart, %CoQ9 was 3%, and in the brain, 2%, and these values did not change with growth. Most CoQ9 was present in the cytosolic fraction, whereas most CoQ10 was in the mitochondrial fraction. There was but minor change in the intracellular distribution of CoQ9 and CoQ10 in rabbit liver between 2 weeks and 7 weeks of age. These results suggest that CoQ9 and CoQ10 may play different roles in their physiological actions as antioxidant or component of the mitochondrial respiratory chain.

Five cases of ectopic liver and a case of accessory lobe of the liver.

Five cases of ectopic liver, two of retro-peritoneal cavity and three of gallbladder, and a case of accessory lobe of the liver, are reported. One of these cases with ectopic liver was accompanied by multiple cysts of the liver and kidney, and biliary microhamartoma, which was observed laparoscopically on the surface of the main liver and histologically proven in the ectopic liver.

Peritoneoscopy as an aid in intravenous injection of indocyanine green (ICG).

Peritoneoscopy as an aid in intravenous injection of indocyanine green (ICG) was clinically evaluated. Hepatic parenchyma was stained after intravenous injection of ICG, while interstitial connective tissue, fatty deposition and hepatoma tissue were not. Regenerative hepatic cell mass including dark reddish patchy marking (Shimada's code No. 7) and semispherical areas of regeneration or nodules (Shimada's code No. 8) was well stained and clearly contrasted. There were some cases of chronic active hepatitis, in which liver surface showed spotty staining at sites expected to become regenerative nodules in the future, in contrast to being judged as "no abnormal findings" peritoneoscopically. On the other hand, periportal reddish marking (Shimada's code No. 4) representing piecemeal or bridging hepatic cell necrosis was not stained.