Effects of quercetin on liver damage in rats with carbon tetrachloride-induced cirrhosis

Flavonoids are reported to exhibit a wide variety of biological effects, including antioxidant and free radical-scavenging activities. Evidence of oxidative reactions is often associated with various chronic disease processes characterized by accumulation of connective tissue. This study was aimed to investigate the protective effects of chronic administration of the flavonoid quercetin (150 mol/kg body wt/day intraperitoneally) in rats with carbon tetrachloride-induced fibrosis. In animals rendered cirrhotic by administration of carbon tetrachloride for 16 weeks, cell necrosis, fibrosis, and inflammatory infiltration were found. Histological abnormalities were accompanied by a higher hepatic content of collagen and thiobarbituric acid-reactive substances. Expression of inducible nitric oxide synthase (iNOS) was significantly increased in the liver. Treatment with quercetin during 3 weeks improved liver histology and reduced collagen content, iNOS expression, and lipid peroxidation. Those effects were associated with an increased total peroxyl radical-trapping antioxidant capacity of liver. We conclude that quercetin is effective in this model of liver damage.     

补肾养肝合剂对四氯化碳诱导大鼠急性肝损伤的防护作用

目的：探讨补肾养肝合剂萃取物对四氯化碳（CCl4）诱导大鼠急性肝损伤之影响。方法：用CCl4诱导急性肝损伤模型，造模前口服补肾养肝合剂萃取物（100，500mg／kg），适时检测血清中ALT、AST活性。结果：补肾养肝合剂可有效降低CCl4诱发之ALT、AST活性；提升肝脏中抗氧化酵素（酶）SOD、GPx、GR活性。结论：结果显示补肾养肝合剂萃取物对CCl4诱导急性肝损伤具有防护作用，其防护CCl4诱导急性肝损伤之机转与提升肝脏内抗氧化酵素（酶）GR、GPx与SOD活性有关。     

Role of L-carnitine in the prevention of acute liver damage induced by carbon tetrachloride in rats

BACKGROUND AND AIM: Lipid peroxidation is the most important mechanism in the pathogenesis of acute liver damage with carbon tetrachloride (CCl4). L-carnitine may prevent lipid peroxidation and thus may protect against liver damage. In the present study we investigated the protective effect of L-carnitine in experimental acute liver damage induced by CCl4. METHODS: Fifty rats were allocated to five equal groups. The first group was the control (group 1), the second group received an intraperitoneal CCl4 injection for 3 days (group 2), and the third group received a 50 mg/kg subcutaneous L-carnitine injection for 4 days, beginning a day before CCl4 injection. The CCl4 injection was continued for 3 days in the concerned group (group 3). Group 4 was given a CCl4 injection for 7 days and group 5 received a 50 mg/kg subcutaneous L-carnitine injection for 8 days, beginning a day before CCl4 injection. This group continued to receive a CCl4 injection for 7 days. Rats in groups 2 and 3 were killed on the fifth day. Rats in groups 1, 4 and 5 were killed on the ninth day. Plasma and liver tissue malondialdehyde (MDA) levels, glutathione peroxidase (GSH-Px) activity and liver enzyme levels were studied. Histopathological investigations were conducted. RESULTS: Liver tissue MDA levels decreased significantly in group 3 compared to group 2 (P<0.001). Liver tissue MDA levels in group 5 decreased significantly in comparison to those of group 4 (P<0.001). Liver tissue GSH-Px activity in group 5 was significantly lower than that in group 4 (P<0.05). There were no significant differences between groups 3 and 4 regarding GSH-Px activity (P>0.05). Steatosis, inflammation and necrosis in group 3 were significantly reduced when compared to group 2 (P<0.01). Fibrosis development was not identified in groups 2 and 3. Steatosis in group 5 was significantly lower than that in group 4 (P<0.05) and there were no significant differences between groups 4 and 5 with regards to inflammation and necrosis (P>0.05). Mild fibrosis development was identified in groups 4 and 5 but the difference between the groups was not significant. CONCLUSION: It appears that L-carnitine has a protective effect in the early stage of experimental acute liver damage induced by CCl4. As the toxic effect or damage continues, its effect lessens.     

Therapeutic effect of melatonin on carbon tetrachloride-induced acute liver injury in rats

The therapeutic effect of melatonin on acute liver injury was examined in rats intoxicated with carbon tetrachloride (CCl4). Melatonin (10, 50, or 100 mg/kg body weight [BW]) was intraperitoneally administered to male Wistar rats 6 hr after intraperitoneal injection of CCl4 (1.6 g/kg BW) at which time an apparent liver injury occurred. This post-melatonin administration dose dependently prevented the progression of liver injury at 24 hr after CCl4 injection, judging from the levels of serum transaminases, indices of liver cell damage. Rats injected with CCl4 alone showed an increase in liver lipid peroxide (LPO) content and a decrease in liver reduced glutathione content at 6 and 24 hr after the injection. The post-melatonin administration dose dependently ameliorated both changes found at 24 hr after CCl4 injection. Rats injected with CCl4 alone showed an increase in liver triglyceride (TG) content and decreases in serum TG concentration and liver tryptophan 2,3-dioxygenase (TDO) activity, a marker of the inhibition of liver protein synthesis by CCl4, at 6 and 24 hr after the injection, and also a decrease in serum albumin concentration at 24 hr. The changes in serum TG, albumin concentration, liver TG content, and TDO activity found at 24 hr after CCl4 injection were not ameliorated by the post-administration of melatonin. The same administration of melatonin dose dependently reduced liver LPO content in CCl4-untreated rats. These results indicate that melatonin exerts a therapeutic effect on CCl4-induced acute liver injury in rats, possibly through its antioxidant action.     

Effect of partially purified NSILA on adenylate cyclase, phosphodiesterase and 3',5'-cyclic AMP in fat cells.

Partially purified, non-suppressible, insulin-like material (NSILA-S) was studied with respect to its effect on the levels of 3',5'-cyclic adenosine monophosphate (cAMP) and its mechanism of action in the control of this nucleotide in rat fat cells. NSILA-S prevents the rise of cAMP in fat cells under the influence of isoproterenol with similar kinetics to insulin. A maximal effect is observed at about 70 ng/ml with a biological activity equivalent to 200 muU/ml of insulin. NSILA-S inhibits norepinephrine-stimulated adenylate cyclase activity in fat cell ghosts and partially purified plasma membrane preparations. At 10 mM Mg2+, the inhibition is characterized by an effect of Vmax without change in affinity towards ATP (apparent KM 30 muM). Similarly there is no observed change in affinity towards Mg2+. With respect to inhibition of norepinephrine-stimulated adenylate cyclase, the dose-response curve of NSILA-S is similar to that already found with intact cells. The effect of norepinephrine is inhibited throughout the dose-response range between 5 X 10(-7) and 5 X 10(-4) M. In contrast to previous observations with insulin in ghosts, NSILA-S inhibits the basal adenylate cyclase activity. Cyclic nucleotide phosphodiesterase activity in homogenates as measured at 1.0 muM substrate is increased by 90% after previous incubation of fat cells with NSILA-S. The study suggests that the anti-lipolytic effect of NSILA-S is mediated by a lowering of cAMP through inhibition of the adenylate cyclase and/or stimulation of the phosphodiesterase system.     

绞股蓝总皂苷对四氯化碳诱导大鼠肝纤维化的防治作用

目的:观察绞股蓝总皂苷对四氯化碳( CCl4)诱导的大鼠肝纤维化的防治作用.方法:采用CCl4诱导的大鼠肝纤维化模型,分为正常组(Z,n=6)、模型组(M,n=8)、绞股蓝总皂苷组(J,n=8)、秋水仙碱(Q,n=8).造模6周末开始给药(股蓝总皂苷200mg/kg体重、秋水仙碱0.1mg/kg体重),给药3周.观察:①大鼠体重、肝脾比值的变化;②血清丙氨酸氨基转移酶(ALT)、门冬氨酸氨基转移酶(AST)、谷氨酰转肽酶(GGT)活性、白蛋白( Alb)、总胆红素(TBil)含量、肝组织羟脯氨酸(Hyp)含量;③肝组织超氧化物歧化酶(SOD)活性及丙二醛(MDA)、还原型谷胱甘肽(GSH)、谷胱甘肽过氧化物酶(GSH-Px)含量;④肝组织病理及胶原沉积情况.结果:①M组大鼠血清ALT、AST、GGT、TBil显著升高,Alb显著降低;J和Q组大鼠血清ALT、AST、GGT、TBil显著下降,Alb显著升高;②M组大鼠肝组织Hyp含量显著升高,J组及Q组大鼠肝组织Hyp含量显著下降;③肝组织HE染色显示:M组大鼠肝细胞脂肪变性,大量纤维结缔组织增生,假小叶形成.J组及Q组大鼠肝细胞脂肪变性减轻,纤维增生减少,少见完整假小叶结构.天狼星红染色显示:M组大鼠肝窦周胶原沉积明显,形成较厚汇管区和中央静脉间的纤维间隔,J组和Q组大鼠肝脏汇管区胶原纤维染较M组明显减轻;④M组大鼠肝组织SOD活性及GSH含量明显降低,MDA含量显著升高.J组大鼠肝组织SOD活性显著提高.结论:绞股蓝总皂苷具有显著抗CCl4诱导的大鼠肝纤维化及氧化损伤的作用.     

The sympathetic nervous system promotes carbon tetrachloride-induced liver cirrhosis in rats by suppressing apoptosis and enhancing the growth kinetics of regenerating hepatocytes

Norepinephrine is considered to possess potent anti-apoptotic action in regenerating hepatocytes. To clarify the role of the sympathetic nervous system in apoptosis that occurs in chronic liver damage and following the promotion of liver cirrhosis, we studied a carbon tetrachloride (CCl₄)-induced liver injury model, using spontaneously hypertensive rats (SHR), Wistar-Kyoto rats (WKY), and chemically sympathectomized WKY. At 24 h after CCl₄ administration, acute damage, characterized by vacuolated hepatocytes in the centrilobular zone, was greater in SHR than in WKY. This vacuolated change in WKY hepatocytes was significantly reduced by chemical sympathectomy with 6-hydroxydopamine (6-OHDA). After 48 h, the acute damage was dramatically improved in each animal, without significant differences between the three groups. In chronic damage after weekly repetition of CCl₄ treatment for 4 weeks, fibrosis was evident in SHR, while in the other groups there was only scant fibrosis in the centrilobular zone. After 8 weeks' repetition of CCl₄, liver cirrhosis was seen only in SHR. The incidence of apoptotic cells in areas of both acute and chronic damage in WKY, detected by terminal deoxynucleotidyl transferase-dUTP nick end labeling, was significantly increased in com-parison with that in SHR, and was further increased by 6-OHDA pretreatment. In contrast, there was significantly greater enhancement of the growth of hepatocytes in SHR than in WKY in both acute and chronic damage. Moreover, hepatocyte growth kinetics in WKY was significantly inhibited after sympathectomy in acute injury, as evidenced by immunohistochemistry for proliferating cell nuclear antigen (PCNA). In vitro, the amount of hepatocellular apoptosis induced by transforming growth factor-β1 was significantly decreased by incubation with norepinephrine. These findings suggest that the anti-apoptotic effect of the sympathetic nervous system increases cell growth kinetics and promotes liver cirrhosis in this animal model. Received: May 22, 2000 / Accepted: September 8, 2000     

The protective effect of pyrroloquinoline quinone and its derivatives against carbon tetrachloride-induced liver injury of rats

Pyrroloquinoline quinone (PQQ) and its derivative, oxazo pyrroloquinoline (OPQ-G), protected rats from experimental liver injury induced by carbon tetrachloride (CCl₄) in vivo. This effect was observed after an intraperitoneal injection of 5 mg/kg PQQ or OPQ-G, which was given twice, 10 min and 1 h before CCl₄ administration. Pyrroloquinoline quinone protected primary cultured rat hepatocytes from CCl₄ toxicity in vitro. This protection was most effective at a concentration of 3 μmol/L PQQ. Pyrroloquinoline quinone derivatives (oxazo pyrroloquinoline, methyl-thioethyl oxazo pyrroloquinoline and PQQ-allylester) also protected the hepatocytes from CCl₄ toxicity. Pyrroloquinoline quinone and its derivatives inhibited the lucigenin-enhanced chemiluminescence from isolated hepatocytes initiated by CCl₄. These results suggest that eliminating free radicals is one of the protective mechanisms of PQQ and its derivatives against CCl₄-induced liver injury.     

Protective effects of BML-111, a lipoxin A4 receptor agonist, on carbon tetrachloride-induced liver injury in mice

Background: Lipoxins (LX) are trihydroxytetraene-containing eicosanoids that display unique anti-inflammatory and pro-resolving actions during various inflammatory conditions, but the pathophysiological significance of LX in liver disorders remains unknown. Methods: In the present study, we used a murine model of carbon tetrachloride (CCl(4))-induced acute liver injury to investigate the effects of LX on the progression of acute liver injury. Results: The results indicated that the lipoxin A(4) receptor (ALX) was upregulated after giving CCl(4). BML-111, a commercially available ALX agonist, effectively protected the liver from CCl(4)-induced injury as evidenced by decreased serum aminotransferase (ALT, AST) levels and improved histological damage. The dampened liver injury was accompanied byreduced malondialdehyde (MDA) content in liver homogenates and decreased concentration of tumor necrosis factor-alpha (TNF-alpha) in the serum. Most interestingly, BML-111 markedly upregulated hepatic heme oxygenase-1 (HO-1) expression in CCl(4)-treated mice, which might provide antioxidative activities in the liver. Conclusion: These data indicate that ALX agonist BML-111 plays a critical protective role in CCl(4)-induced acute liver injury through limiting the inflammatory response and promoting antioxidative protein expression.     

The effects of dichloroacetate on liver damage and circulating fuels in rats exposed to carbon tetrachloride

Abstract It has been known that carbon tetrachloride-induced liver damage in starved rats is ameliorated simply by restoration of feeding. An analogue of dichloroacetate has been reported to ameliorate carbon tetrachloride-induced liver damage, and dichloroacetate has been shown to have a variety of effects on fuel metabolism. We investigated simultaneously the effects of dichloroacetate on liver damage and on circulating fuels in rats exposed to carbon tetrachloride. The effects of carbon tetrachloride varied with the rat's condition. In starved rats, the liver damage was more severe, and serum ketone body concentration decreased. In non-starved rats, the liver damage was not as severe and the serum ketone body concentration increased. The administration of dichloroacetate ameliorated liver damage both in starved and in non-starved rats given carbon tetrachloride: the administration of dichloroacetate protected from the liver damage particularly in starved rats. There were associated changes in the concentractions of circulating fuels. When the pyruvate-lowering effect of dichloroacetate was diminished in carbon tetrachloride-injected, starved rats, the alanine aminotransferase-lowering effect of dichloroacetate was also diminished. We propose that dichloroacetate's effect on fuel metabolism may produce a hepato-protective effect.     

The protective role of Hepatopoietin Cn on liver injury induced by carbon tetrachloride in rats*

Background: Hepatopoietin Cn (HPPCn) is a member of the leucine‐rich acidic nuclear protein family (LANP), and studies of partially hepatectomized (PH) mice show that levels of HPPCn mRNA increase following liver injury. Furthermore, the recombinant human protein (rhHPPCn) was shown to stimulate hepatic DNA synthesis and activate signaling pathways involved in hepatocyte proliferation in vitro and in vivo. Aim: The aim of the study was to evaluate the protective effect of rhHPPCn on liver injury and fibrosis induced by carbon tetrachloride (CCl4) injection. Methods: Wistar rats weighing 200 g were given a single and repeated intraperitoneal injections of CCl4. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activity in rat serum were measured using biochemical assay. Hepatic hydroxyproline (Hyp) level was determined in the hydrolysates of liver samples. Immunostaining and Masson's trichrome staining were conducted to evaluate hepatocyte proliferation and fibrosis. Results: The results showed that exogenous rhHPPCn could alleviate hepatocyte necrosis and protect the liver from the development of fibrotic lesions by proliferation stimulation. Additionally, HPPCn could reduce ALT/AST levels in rat serum following single and repeated CCl4 injection. Conclusion: It was suggested that HPPCn could protect hepatocytes from injury induced by CCl4 as a proliferation stimulator.     

Effects of carbamylcholine and ionophore A-23187 on cyclic 3',5'-AMP and cyclic 3',5'-GMP accumulation in dog-thyroid slices.

Carbamylcholine and acetylcholine through a muscarinic type of receptor, KCl, ionophore A-23187 and NaF increased cyclic GMP accumulation in dog-thyroid slices. These effects were abolished in calcium-depleted slices, which findings confirm that Ca2+ is required for cyclic GMP accumulation. All these agents depressed the accumulation of cyclic AMP in TSH-stimulated slices. KCl and NaF depressed cyclic AMP accumulation in TSH-treated slices even when they had been depleted of Ca2+. This suggests a cyclic GMP- and Ca2+-independent mechanism. The absence of inhibition of the effects of the ionophore, NaF and KCl in the presence of atropine suggests that these drugs do not act by inducing the release of acetylcholine in the slices. The effects of carbamylcholine and ionophore A-23187 on cyclic GMP accumulation and protein iodination were reversible; the inhibitions of TSH-induced cyclic AMP accumulation and secretion were non-competitive and were not accompanied by a depression of ATP levels. All these effects were greatly decreased in the absence of extracellular Ca2+. These data suggest that carbamylcholine and ionophore A-23187 act mainly by increasing the influx of extracellular Ca2+ in thyroid cells. However, the persistence of some carbamylcholine effect in the absence of Ca2+ in the medium suggests that this agent may also trigger the release of Ca2+ from an intrafollicular pool. The kinetics of action of carbamylcholine are compatible with a role of cyclic GMP in the inhibition of cyclic AMP accumulation. However, with the ionophore, the depression of cyclic AMP accumulation was much longer than the rise of cyclic GMP, which suggests a mechanism independent of cyclic GMP.     

The pathology of liver injury induced by the chronic administration of alcohol and 'low-dose' carbon tetrachloride in Porton rats

We have previously established a model for micronodular cirrhosis by feeding Wistar rats alcohol, in the Lieber–DeCarli liquid diet, and exposing them to ‘low-dose’ carbon tetrachloride (CCl₄) vapour for 10 weeks. This study reports the spectrum of liver pathology seen in male Porton rats exposed to ‘low-dose’ CCl₄ vapour 5 nights/week, 6 h/night while being fed alcohol (300 kcal/L) in the Lieber-DeCarli diet. Micronodular cirrhosis developed in all animals after 5–7 weeks of treatment. The simultaneous administration of silymarin, a putative hepatoprotective agent, in the liquid diet, did not alleviate or prevent the chronic liver injury. The histopathological features of the liver injury are described, with particular emphasis on the presence of small epithelial cells (‘progenitor or stem cell'), which appear to be playing a role in liver regeneration.     

Hepatoprotective effect of Cichorium intybus L.,a traditional Uighur medicine,against carbon tetrachloride-induced hepatic fibrosis in rats

AIM:To investigate the hepatoprotective effect of a Cichorium intybus L.extract(CIE)on CCl4-induced hepatic fibrosis in rats.METHODS:Seventy-two male Wistar albino rats were randomly divided into six groups of twelve rats each.The normal control group was allowed free access to food and water.Liver injury was performed in the remaining five groups with an i.p.injection of a 1.0mL/kg CCl4 and olive oil(2:3 v/v)mixture,twice weekly for 8 weeks.All rats,with the exception of the injury model group,were intragastrically(i.g.,)administered quantum satis(q.s.)dosages[CIE group:6,18,and 54 mg/kg,respectively;Fu Fang Bie Jia Ruan Gan Pian(FFBJRGP)group:780 mg/kg].The oral administration of different drugs was performed on the day before CCl4 administration and subsequently once per day for8 wk.The serum levels of aspartate aminotransferase(AST),alanine aminotransferase(ALT),hexadecenoic acid(HA),laminin(LN),hydroxyproline(Hyp),and glutathione(GSH),malondialdehyde(MDA)and superoxide dismutase(SOD)in the rat livers were measured.Histopathological changes in the liver were assessed for each group using HE staining and a Masson Trichrome examination.The expression of transforming growth factor-β1(TGF-β1)andα-smooth muscle actin(α-SMA)was examined by immunohistochemical analysis.RESULTS:CIE at oral doses of 6,18,and 54 g/kg per day showed a significant hepatoprotective effect,especially at a dose of 54 g/kg per day.CIE doses reduced the levels of AST(149.04±34.44,P<0.01),ALT(100.72±27.19,P<0.01),HA(548.50±65.09,P<0.01),LN(28.69±3.32,P<0.01)and Hyp(263.33±75.82,P<0.01).With regards to hepatoprotective activity,the CIE dose of 54 g/kg per day produced the largest significant effect by increasing GSH(3.11±0.81),SOD(269.98±33.77,P<0.01)and reducing MDA(2.76±0.51,P<0.01)levels in the liver.The expressions of TGF-β1 andα-SMA were measured by immunohistology and found to be significantly reduced by CIE in a dose-dependent manner.     

Antioxidant and hepatoprotective effects of Ginkgo biloba phytosomes in carbon tetrachloride-induced liver injury in rodents.

AIM: The protective effects of Ginkgo biloba phytosomes (GBP) on carbon tetrachloride (CCl4)-induced hepatotoxicity and the probable mechanism(s) involved in this protection were investigated in rats. METHODS: Liver damage was induced in Wistar rats by administering a 1:1 (v/v) mixture of CCl4 and olive oil (1 ml/kg, i.p.) once daily for 7 days. GBP at 25 mg/kg and 50 mg/kg, i.p. and reference drug silymarin (200 mg/kg, p.o.) were administered for 10 days to CCl4-treated rats, this treatment beginning 3 days prior to the commencement of CCl4 administration. The degree of protection was evaluated by determining the marker enzymes (SGOT, SGPT and SALP), albumin (Alb) and total proteins (TP). Further, the effects of GBP on lipid peroxidation (LPO), glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and glutathione reductase (GR) were estimated in liver homogenates to evaluate antioxidant activity. RESULTS: GBP (25 and 50 mg/kg) and silymarin elicited significant hepatoprotective activity by decreasing the activities of serum marker enzymes and lipid peroxidation and elevated the levels of GSH, SOD, CAT, GPX, GR, Alb and TP in a dose-dependent manner. CONCLUSION: The present findings indicate that the hepatoprotective effects of GBP against CCl4-induced oxidative damage may be due to its antioxidant and free radical-scavenging activity.     

Plasma kallikrein clearance by the liver of chronic carbon tetrachloride-treated rats

Abstract We have previously reported that the endocytosis of rat plasma kallikrein (RPK) by hepatocytes is a calcium-independent and beta-galactoside-dependent mechanism. We now report the clearance of RPK by the liver of four groups of rats: normal, inflamed (48 h ex-turpentine) and two groups chronically treated with CCl₄ (52 mg/kg per week, intragastrically, for 9-12 weeks). Each liver was isolated, exsanguinated and perfused at 37°C with 30 mL of BSA-Krebs-Henseleit-bicarbonate medium containing 10 nmol/L RPK. Although all rats received the same mild CCl₄ treatment, the liver histology showed that they evolved either to severe hepatitis (serum alanine aminotransferase [ALT] 4852 ± 885 U/L, parenchymatous necrosis in the perivenous region) or to compensated cirrhosis (serum ALT 209 ± 42 U/L, vigorous fibrous encircling regeneration nodules); neither jaundice nor ascites was noted. The results show that serum albumin was not altered among the groups and that: the acute-phase response by itself (inflamed group) increased RPK clearance rate (3.01 ± 0.59 mL/min) as compared with the normal group (1.85 ± 0.14 mL/min); the CCl₄ treatment, although induced an acute-phase response, decreased ( P < 0.01) RPK clearance rates (0.80 ± 0.11 mL/min hepatitis group and 0.98 ± 0.10 mL/min cirrhosis group). These findings suggest that the hepatic clearance rate of plasma kallikrein is an early indicator of liver injury.     

The effect of carbon tetrachloride and ultraviolet radiation on dolichol levels in liver cells isolated from 3- and 24-month-old male Sprague-Dawley rats

Dolichol (D) is a long-chain polyprenoid broadly distributed in the cell membranes, possibly endowed with a free-radical scavenging activity, whose concentration in tissues increases with increasing age. No enzyme pathway for D degradation has been discovered. In order to test the hypothesis that D might undergo a non-enzymatic free-radical mediated decomposition the effects of a xenobiotic agent (carbon tetrachloride, CCl(4)) and ultraviolet-B (UV-B) radiation on D levels were studied in liver cells isolated from male ad libitum fed Sprague-Dawley rats aged 3 or 24 months. Liver cells (90 mg/ml) were incubated in sealed flasks (6 ml cell suspension each) for 0, 5, 10 and 20 min after the addition of 25, 50 or 200 microl CCl(4) in the central well. 50 ml of a 6 mg/ml liver cell suspension were poured in a 120 cm(2) Petri dish and the sediment liver cell monolayer was exposed to UVB radiation for 0, 5, 10, 20 and 40 min. At the given time, cells were taken and D was extracted and assayed by the HPLC procedure. D levels were remarkably higher in older than in younger cells as expected ( P < 0.001). Treatment with CCl(4) and UVB caused a highly significant decrease in D ( P < 0.001) whose percentage was larger in younger than in older cells. The conclusions are that free-radicals generated either by chemical or by physical agents cause a very rapid depletion of D in liver cells, and that the effect of the free radical attack on D decomposition may be lower percentage wise in older than in younger cells, which might account at least in part for the accumulation of D in older tissues.