Prevention of Carbon Tetrachloride-Induced Lipid Peroxidation in Liver Microsomes from Dehydroepiandrosterone-Pretreated Rats

Dehydroepiandrosterone (DHEA), a lipid soluble steroid, administered to rats (100 mg/kg b.wt) by a single intraperitoneal injection, increases to twice its normal level in the liver microsomes. Microsomes so enriched become resistant to lipid peroxidation induced by incubation with carbon tetrachloride in the presence of a NADPH-regenerating system: also the lipid peroxidation-dependent inactivation of glucose-6-phosphatase and gamma-glutamyl transpetidase due to the haloalkane are prevented. Noteworthy, the liver microsomal drug-metabolizing enzymes and in particular the catalytic activity of cytochrome P₄₅₀IIE1, responsible for the CCl₄-activation, are not impaired by the supplementation with the steroid. Consistently, in DHEA-pretreated microsomes the protein covalent binding of the trichloromethyl radical (CCl₃°), is similar to that of not supplemented microsomes treated with CCl₄. It thus seems likely that DHEA protects liver microsomes from oxidative damage induced by carbon tetrachloride through its own antioxidant properties rather than inhibiting the metabolism of the toxin.     

Pharmacological properties of dibenzo[a,c]cyclooctene derivatives isolated from Fructus Schizandrae chinensis III. Inhibitory effects on carbon tetrachloride-induced lipid peroxidation,metabolism and covalent binding of carbon tetrachloride to lipids

Fructus Schizandrae, a traditional Chinese tonic, has been shown to lower the elevated serum glutamic pyruvic transaminase (SGPT) levels of patients with chronic viral hepatitis and several of its components decrease the hepatotoxicity of carbon tetrachloride (CCl₄) in animals. This paper deals with the mechanism of protection against CCl₄-hepatotoxicity of these compounds as well as of DDB, a synthetic analogue of Schizandrin (Sin) C. Of the seven components, Sin B and C, Schizandrol (Sol) B, Schizandrer (Ser) A and B, as well as dimethyl-4,4′-dimethoxy-5,6,5′,6′-dimethylenedioxy-biphenyl-2,2′-dicarboxylate (DDB) were shown to inhibit CCl₄-induced lipid peroxidation and [¹⁴C]Cl₄ covalent binding to lipids of liver microsomes from phenobarbital(PB)-treated mice. The compounds also decreased carbon monoxide (CO) production and cofactor (NADPH, oxygen) utilization during CCl₄ metabolization by liver microsomes. It may be postulated, therefore, that the hepatoprotective effect of certain components isolated from Fructus Schizandrae as well as DDB is due to their inhibitory effect on CCl₄-induced lipid peroxidation and the binding of CCl₄-metabolites to lipids of liver microsomes.     

Lipoperoxidation after carbon tetrachloride poisoning in rats previously treated with antioxidants

Propyl gallate (PG), reduced glutathione (GSH) and N,N′-diphenyl-p-phenylenediamine (DPPD), administered to rats prior to carbon tetrachloride, protect against hepatic fat infiltration until the fourth hour after poisoning. This effect does not seem to be mediated by a block in lipid mobilization from depot fat.A preliminary treatment with DPPD succeeds in inhibiting the double bond shifting in liver microsomal lipids within 30 min after dosing with CCl₄. The early peroxidative alteration occurs at the normal rate after the administration of either GSH or PG. The amount of lipid-bound radiocarbon and of ¹⁴CO₂ exhaled within 2 h after intragastric ¹⁴C-labelled carbon tetrachloride is not affected by the preliminary protection with the antioxidants.CCl₄ metabolites and/or lipoperoxides impair the in vitro combination of serum apoprotein with lipid. No changes are observed when lipoperoxidation is inhibited by antioxidants.These findings are interpreted as a support for the hypothesis that the possible contribution given by the enhancement of lipid peroxidation to the pathogenesis of CCl₄-induced fatty liver could depend on further structural and functional alterations occurring in the cytoplasmic environment of hepatocytes rather than the early radical attack onto the unsaturated lipids of liver microsomes. The functional integrity and the supply of the protein carrier for the triglyceride secretion mechanisms could be considered a target of the hepatotoxic action of CCl₄, at the molecular level.     

Factors Influencing the Formation of the Carbon Dioxide Radical Anion (CO2−) Spin Adduct of Pbn in the Rat Liver Metabolism of Halocarbons

Spin trapping techniques have been used to detect free radicals generated from the in vitro metabolism by rat liver microsomes of carbon tetrachloride (CCl₄) and bromotrichloromethane (BrCCI) under conditions of varying oxygen tension and pH. Dispersions of rat liver microsomes incubated with ¹²CCl₄, ¹³CCl₄ or Br¹²CCl₃, α-phenyl-tert-butyl nitrone (PBN) and NADPH/NADH in a phosphate buffer varying in pH from 6.6 to 8.0 under varying oxygen tensions produced various amounts of four different PBN adducts: PBN-CCl₃, PBN-L, PBN-OL and PBN-CO^?₂ where L is a carbon-centered lipid type radical and LO is an oxygen-centered lipid type radical. The relative amount of PEN-CO; increases with the absence of oxygen. With the use of ³¹P-NMR in vivo spectroscopy it was possible to detect a pH change from 7.4 to 6.8 in the livers of rats treated with CCl₄, or BrCCl₃. These results suggest that halocarbon metabolism in biological systems may depend on both oxygen tension as well as pH.     

Identification of dichloromethyl carbene as a metabolite of carbon tetrachloride

Although indirect evidence has suggested that liver microsomal cytochrome P-450 can reductively dehalogenate several compounds to carbene metabolites, there has been no direct proof for the formation of these reactive species. We report in this paper that carbenes can be chemically trapped and identified as metabolites. For example, 1,1-dichloro-2,2,3,3-tetramethylcyclopropane was identified as a metabolite by gas chromatography mass spectrometry when carbon tetrachloride (CCl₄) was incubated anaerobically with rat liver microsomes, NADPH and 2,3-dimethyl-2-butene. The reaction required NADPH and was inhibited by carbon monoxide. These findings show that cytochrome P-450 in rat liver microsomes can reductively metabolize CCl₄ to dichloromethyl carbene (:CCl₂) which can be trapped with 2,3-dimethyl-2-butene to form 1,1-dichloro-2,2,3,3-tetramethylcyclopropane. A similar approach may be used for the identification of carbene metabolites of other compounds.     

Propolis protects CYP 2E1 enzymatic activity and oxidative stress induced by carbon tetrachloride

Induction of CYP 2E1 by carbon tetrachloride (CCl₄) is one of the central pathways by which CCl₄ generates oxidative stress in hepatocytes. Experimental liver injury was induced in rats by CCl₄ to determine toxicological actions on CYP 2E1 by microsomal drug metabolizing enzymes. In this report, ethanolic extract of propolis at a dose of 200 mg/kg (po) was used after 24 h of toxicant administration to validate its protective potential. Intraperitoneal injection of CCl₄ (1.5 ml/kg) induced hepatotoxicity after 24 h of its administration that was associated with elevated malonyldialdehyde (index of lipid peroxidation), lactate dehydrogenase and γ-glutamyl transpeptidase release (index of a cytotoxic effect). Hepatic microsomal drug metabolizing enzymes of CYP 2E1 showed sharp depletion as assessed by estimating aniline hydroxylase and amidopyrine N-demethylase activity after CCl₄ exposure. Toxic effect of CCl₄ was evident on CYP 2E1 activity by increased hexobarbitone induced sleep time and bromosulphalein retention. Propolis extract showed significant improvement in the activity of both enzymes and suppressed toxicant induced increase in sleep time and bromosulphalein retention. Choleretic activity of liver did not show any sign of toxicity after propolis treatment at a dose of 200 mg/kg (id). Histopathological evaluation of the liver revealed that propolis reduced the incidence of liver lesions including hepatocyte swelling and lymphocytic infiltrations induced by CCl₄. Electron microscopic observations also showed improvement in ultrastructure of liver and substantiated recovery in biochemical parameters. Protective activity of propolis at 200 mg/kg dose was statistically compared with positive control silymarin (50 mg/kg, po), a known hepatoprotective drug seems to be better in preventing hepatic CYP 2E1 activity deviated by CCl₄. These results lead us to speculate that propolis may play hepatoprotective role via improved CYP 2E1 activity and reduced oxidative stress in living system.     

S-allyl cysteine prevents CCl4-induced acute liver injury in rats

Aged garlic extract (AGE) possesses multiple biological activities. We evaluated the protective effect of S-allyl cysteine (SAC), one of the organosulfur compounds of AGE, against carbon tetrachloride (CCl₄)-induced acute liver injury in rats. SAC was administrated intraperitoneally (50–200 mg/kg). SAC significantly suppressed the increases of plasma ALT and LDH levels. SAC also attenuated histological liver damage. CCl₄ administration induced lipid peroxidation accompanied by increases in the plasma malondialdehyde and hepatic 4-hydroxy-2-nonenal levels, and SAC dose-dependently attenuated these increases. The hepatic total level of hydroxyoctadecadienoic acid (HODE), a new oxidative stress biomarker, was closely correlated with the amount of liver damage. These results suggest that SAC decreased CCl₄-induced liver injury by attenuation of oxidative stress, and may be a better therapeutic tool for chronic liver disease.     

Protective Effect of Cornuside against Carbon Tetrachloride-Induced Acute Hepatic Injury

This study elucidated the effects of cornuside on carbon tetrachloride (CCl₄)-induced hepatotoxicity. Rats were treated intraperitoneally with 0.5 mL/kg of CCl₄. Sixteen h after CCl₄ treatment, the levels of serum aminotransferases, tumor necrosis factor-α (TNF-α), and lipid peroxidation were significantly elevated, whereas the hepatic antioxidative enzyme activities were decreased. These changes were attenuated by cornuside. Histological studies also indicated that cornuside inhibited CCl₄-induced liver damage. Furthermore, the contents of hepatic nitrite, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) were elevated after CCl₄ treatment, while cytochrome P450 2E1 (CYP2E1) expression was suppressed. Cornuside treatment inhibited the formation of liver nitrite, and reduced the overexpression of iNOS and COX-2 proteins, but restored the liver CYP2E1 content as compared with the CCl₄-treated rats. Our data indicate that cornuside protects the liver from CCl₄-induced acute hepatotoxicity, perhaps due to its ability to restore the CYP2E1 function and suppress inflammatory responses, in combination with its capacity to reduce oxidative stress.     

Investigation of the Hepatoprotective Effects of Sesame (Sesamum indicum L.) in Carbon Tetrachloride-Induced Liver Toxicity

More than 600 chemicals can cause damage in liver, one of which is carbon tetrachloride (CCl₄). Hepatoprotective agents could prevent tissue damage and reduce morbidity and mortality rates; such agents may include alternative or folkloric treatments. We investigated sesame (Sesamum indicum L.) for its hepatoprotective effect in CCl₄-induced experimental liver damage. To this end, 0.8 mg/kg of sesame fixed oil was provided intraperitoneally to rats whose livers were damaged by CCl₄. Tissue and blood samples were taken at the end of the experiments and evaluated histologically and biochemically. Ballooning degenerations and an increase in lipid droplets in liver parenchyma and increases in serum alanine transaminase, aspartate transaminase, and bilirubin were found in the CCl₄ group. Biochemical and histopathological findings in the sesame fixed oil treated group were not significantly different from the CCl₄ group. Sesame did not show a hepatoprotective effect in CCl₄-induced liver toxicity.     

3-Alkynyl selenophene protects against carbon-tetrachloride-induced and 2-nitropropane-induced hepatic damage in rats

The aim of this study was to investigate the protective effect of 3-alkynyl selenophene (3-ASP) on acute liver injury induced by carbon tetrachloride (CCl₄) and 2-nitropropane (2-NP) in rats. On the first day of treatment, the animals received 3-ASP (25 mg/kg, p.o.). On the second day, the rats received CCl₄ (1 mg/kg, i.p.) or 2-NP (100 mg/kg, p.o.). Twenty-four hours after CCl₄ or 2-NP administration, the animals were euthanized, and their plasma and liver were removed for biochemical and histological analyses. The histological analysis revealed extensive injury in the liver of CCl₄-exposed and 2-NP-exposed rats, which was attenuated by 3-ASP. 3-ASP significantly attenuated (1) the increase in plasmatic aspartate and alanine aminotransferase activities and lipid peroxidation levels induced by CCl₄ and 2-NP; (2) the inhibition of δ-aminolevulinic dehydratase activity caused by 2-NP; and (3) the decrease in ascorbic acid (AA) levels and catalase (CAT) activity caused by CCl₄. AA levels and CAT activity remained unaltered in the liver of rats exposed to 2-NP. The protective effect of 3-ASP on acute liver injury induced by CCl₄ and 2-NP in rats was demonstrated.     

Spin-trapping of the trichloromethyl radical produced during enzymic NADPH oxidation in the presence of carbon tetrachloride or bromotrichloromethane

Utilizing the spin-trapping agent phenyl-t-butyl nitrone, a free radical has been detected which is produced from carbon tetrachloride or bromotrichloromethane during the enzymic oxidation of NADPH by rat liver microsomes. The presence of NADPH is obligatory for generation of the radical.The formation of the trichloromethyl radical-phenyl-t-butyl nitrone adduct is an enzymic process, as evidenced by the inhibition of its formation in systems containing heated microsomes and in systems containing p-hydroxymercuribenzoate. A computer-simulated ESR spectrum for the trichloromethyl adduct of phenyl-t-butyl nitrone can reproduce the essential features of the spectrum of the spin-trapped radical produced enzymically from CCl₄. A mechanism is proposed for the formation of the trichloromethyl radical from CCl₄ or BrCCl₃.     

Hepatic calcium-binding protein regucalcin is released into the serum of rats administered orally carbon tetrachloride

The change in calcium-binding protein regucalcin, mainly localized in liver, in the liver and serum of rats received a single oral administration of carbon tetrachloride (50%; 1.0 ml/100 g body weight) was investigated. The change of regucalcin mRNA levels in the liver was analyzed by Northern blotting using liver regucalcin cDNA (0.6 kb). At 10 and 24 h after the administration, liver regucalcin mRNA levels were reduced markedly. Moreover, regucalcin concentration in the liver and serum was estimated by enzyme-linked immunoadsorbent assay (ELISA) with rabbit-anti-regucalcin IgG. Administration of carbon tetrachloride (CCl₄) induced a significant decrease in liver regucalcin concentration and a corresponding elevation of serum regucalcin concentration at 24 h after the administration. An appreciable increase in serum regucalcin concentration was seen at 2 h after the administration. Meanwhile, serum transaminases (GOT and GPT) activities were significantly increased by CCl₄ administration, indicating that liver injury is induced. The present study demonstrates that hepatic regucalcin is released into the serum of rats administered orally CCl₄, suggesting that the estimation of serum regucalcin is a useful tool for diagnosis of liver injury.     

Antioxidant and hepatoprotective activities of low molecular weight sulfated polysaccharide from Laminaria japonica

A low molecular weight sulfated polysaccharide (LMWF) was prepared from Laminaria japonica by mild acid hydrolysis. The antioxidant activity of LMWF in vitro was studied using three kinds of oxygen free radical systems. LMWF had effective scavenging abilities on superoxide radical, hydroxyl radical and hypochlorous acid directly in vitro. The hepatoprotective effect of LMWF was studied using two acute liver injury mice models induced by carbon tetrachloride (CCl₄) and D-galactosamine (D-GalN). Addition of LMWF significantly lowered the content of serum malonaldehyde and markedly increased the activities of superoxide dismutase and glutathione peroxidase, compared with the model groups in both kinds of liver injury mice. Moreover, administration of LMWF significantly inhibited the elevation of glutamate pyruvate transaminase induced by CCl₄ and D-GalN in mice. The results suggest that the antioxidant activity of LMWF plays an important role in its hepatoprotective effect in the liver injury mice induced by CCl₄ and D-GalN.     

Podophyllum hexandrum aqueous extract as a potential free radical scavenger

Abstract

The present study was undertaken to evaluate the effect of the aqueous extract of Podophyllum hexandrum against free radical-mediated damage and also explore its anticancer activity. The extract exhibited significant activity in scavenging 1, 1-diphenyl-2-picryl-hydrazyl radicals, ^?OH radical-mediated DNA damage, and lipid peroxide production in rat liver microsomes. The extract was also tested for its reducing abilities. The activity of liver marker enzymes and antioxidant defense enzymes in rat liver homogenate was assessed in control and carbon tetrachloride (CCl₄)-treated animals. It was observed that CCl₄-induced changes viz., increases in the activities of aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase, a decrease in reduced glutathione as well as decreases in the activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glutathione-S-transferase. All these parameters showed reversal when pretreated with aqueous extract of P. hexandrum. Podophylotoxin and etoposide are the two known anticancer agents derived from P. hexandrum and interestingly the aqueous extract of P. hexandrum showed a typical DNA ladder formation in HL-60 cells confirming its role as an inducer of apoptosis. The results obtained suggest that the plant extract exhibits inhibition of and free radical production and lipid peroxidation, increase in antioxidant enzyme activities, revealing its antioxidant properties, and is also able to show potent anticancer activity as depicted by its ability to cause fragmentation of DNA.     

Hepatoprotective,Immunomodulatory, and Anti-inflammatory Activities of Selenocystine in Experimental Liver Injury of Rats

The study was evaluated to investigate the efficacy of selenocystine (CysSeSeCys), a well-known organoselenium compound, on the prevention of carbon tetrachloride (CCl₄)-induced acute hepatic injury in Wistar rats. Forty healthy male Wistar rats were utilized in this study. Acute hepatotoxicity was induced by CCl₄ intoxication in rats. Serum biological analysis, oxidative stress, immune parameters, and gene expression of COX-2 and CYP2E1 were carried out. Pretreatment of CysSeSeCys prior to CCl₄ administration significantly prevented an increase in serum hepatic enzymatic activities. In addition, pretreatment of CysSeSeCys significantly prevented the formation of ROS, MDA, depletion of glutathione, and alteration of antioxidant enzyme activities in the liver of CCl₄-intoxicated rats. This study also revealed that pretreatment with CysSeSeCys normalized the levels of interleukin 6 and10, IgG, and CD4 cell count. Pretreatment of CysSeSeCys significantly reversed COX-2 inflammatory response and the upregulation of CYP2E1 expression as well. Histopathological changes induced by CCl₄ were also significantly attenuated by CysSeSeCys pretreatment. CysSeSeCys has a potent hepatoprotective effect on CCl₄-induced liver injury in rats through its antioxidative, immunomodulatory and anti-inflammatory activity.     

Exopolysaccharide-peptide complex from oyster mushroom (Pleurotus ostreatus) protects against hepatotoxicity in rats

Liver damage involves oxidative stress and a progression from chronic hepatitis to hepatocellular carcinoma (HCC). The increased incidence of liver disease in Egypt and other countries in the last decade, coupled with poor prognosis, justify the critical need to introduce alternative chemopreventive agents that may protect against liver damage. The aim of this study was to evaluate the efficacy of exopolysaccharide-peptide (PSP) complex extracted from Pleurotus ostreatus as a hepatoprotective agent against diethylnitrosamine (DEN)/carbon tetrachloride (CCL₄)-induced hepatocellular damage in rats. The levels of liver injury markers (ALT, AST and ALP) were substantially increased following DEN/CCl₄ treatment. DEN/CCl₄ - induced oxidative stress was confirmed by elevated levels of lipid peroxidation and decreased levels of superoxide dismutase, glutathione-S-transferase, and reduced glutathione. PSP reversed these alterations in the liver and serum, and provided protection evidenced by reversal of histopathological changes in the liver. The present study demonstrated that PSP extract from P. ostreatus exhibited hepatoprotective and antioxidant effects against DEN/CCl₄-induced hepatocellular damage in rats. Given the high prevalence of HCV-related liver damage in Egypt, our results suggest further clinical evaluation of P. ostreatus extracts and their potential hepatoprotective effects in patients with liver disease.     

Sulforaphane ameliorates ethanol plus carbon tetrachloride-induced liver fibrosis in mice through the Nrf2-mediated antioxidant response and acetaldehyde metabolization with inhibition of the LPS/TLR4 signaling pathway

Alcoholic liver disease (ALD)-related fibrosis results from a variety of mechanisms including the accumulation of acetaldehyde, reactive oxygen species, and hepatic overload of endogenous lipopolysaccharide (LPS). Alcohol cessation is the therapeutic mainstay for patients with all stages of ALD, whereas pharmacological strategies for liver fibrosis have not been established. Sulforaphane, a phytochemical found in cruciferous vegetables, activates nuclear factor erythroid 2-related factor 2 (Nrf2) and exerts anticancer, antidiabetic, and antimicrobial effects; however, few studies investigated its efficacy in the development of ALD-related fibrosis. Herein, we investigated the effect of sulforaphane on acetaldehyde metabolism and liver fibrosis in HepaRG and LX-2 cells, human hepatoma and hepatic stellate cell lines, respectively, as well as in a mouse model of alcoholic liver fibrosis induced by ethanol plus carbon tetrachloride (EtOH/CCl₄). Sulforaphane treatment induced the activity of acetaldehyde-metabolizing mitochondrial aldehyde dehydrogenase in HepaRG cells and suppressed the acetaldehyde-induced proliferation and profibrogenic activity in LX-2 cells with upregulation of Nrf2-regulated antioxidant genes, including HMOX1, NQO1, and GSTM3. Moreover, sulforaphane attenuated the LPS/toll-like receptor 4-mediated sensitization to transforming growth factor-β with downregulation of NADPH oxidase 1 (NOX1) and NOX4. In EtOH/CCl₄-treated mice, oral sulforaphane administration augmented hepatic acetaldehyde metabolism. Additionally, sulforaphane significantly inhibited Kupffer cell infiltration and fibrosis, decreased fat accumulation and lipid peroxidation, and induced Nrf2-regulated antioxidant response genes in EtOH/CCl₄-treated mice. Furthermore, sulforaphane treatment blunted hepatic exposure of gut-derived LPS and suppressed hepatic toll-like receptor 4 signaling pathway. Taken together, these results suggest sulforaphane as a novel therapeutic strategy in ALD-related liver fibrosis.     

Effects of indoleamine 2,3‐dioxygenases in carbon tetrachloride‐induced hepatitis model of rats

Indoleamine 2,3‐dioxygenase (IDO) converts tryptophan to l ‐kynurenine, and it is noted as a relevant molecule in promoting tolerance and suppressing adaptive immunity. In this study, to investigate the effects of IDO in carbon tetrachloride (CCl₄)–induced hepatitis model, the levels of IDO enzymic activities in the mock group, the control group and the 1‐methyl‐d ‐tryptophan (1‐MT)–treated group were confirmed by determination of l ‐kynurenine concentrations. Serum alanine aminotransferase levels in 1‐MT‐treated rats after CCl₄ injection significantly increased compared with those in mock and control groups. In CCl₄‐induced hepatitis models, tumour necrosis factor‐α (TNF‐α) is critical in the development of liver injury. The mRNA expression and secretion levels of TNF‐α in the liver from 1‐MT‐treated rats were more enhanced compared with those in the mock and the control groups. Moreover, the levels of cytokine and chemokine from mock, control group and 1‐MT‐treated rats after treated with CCl₄ were analyzed by ELISA, and the level of interleukin‐6 was found to increase in 1‐MT‐treated rats. It was concluded that the deficiency of IDO exacerbated liver injury in CCl₄‐induced hepatitis and its effect may be connected with TNF‐α and interleukin‐6. Copyright © 2012 John Wiley & Sons, Ltd.     

Protective effect of bixin on carbon tetrachloride-induced hepatotoxicity in rats

Background

The liver is an important organ for its ability to transform xenobiotics, making the liver tissue a prime target for toxic substances. The carotenoid bixin present in annatto is an antioxidant that can protect cells and tissues against the deleterious effects of free radicals. In this study, we evaluated the protective effect of bixin on liver damage induced by carbon tetrachloride (CCl₄) in rats.

Results

The animals were divided into four groups with six rats in each group. CCl₄ (0.125 mL kg^-1 body wt.) was injected intraperitoneally, and bixin (5.0 mg kg^-1 body wt.) was given by gavage 7 days before the CCl₄ injection. Bixin prevented the liver damage caused by CCl₄, as noted by the significant decrease in serum aminotransferases release. Bixin protected the liver against the oxidizing effects of CCl₄ by preventing a decrease in glutathione reductase activity and the levels of reduced glutathione and NADPH. The peroxidation of membrane lipids and histopathological damage of the liver was significantly prevented by bixin treatment.

Conclusion

Therefore, we can conclude that the protective effect of bixin against hepatotoxicity induced by CCl₄ is related to the antioxidant activity of the compound.