CCl4-induced Oxidative Damage In Rats Research Articles

Antioxidant and Hepatoprotective Effects of L-Glu and NAC against CCl4-induced Oxidative Damage in Rats. Biochemical and Histopathological Evaluation

Background: The imbalance between free radical formation and antioxidant defence leads to the development of oxidative stress. The search for substances that would mitigate or prevent the effects of oxidative stress remains relevant. Objective: Our goal was to compare the antioxidant and mitigation effects of L-glutamic acid (LGlu) and N-acetylcysteine (NAC) alone or in combination using a battery of biomarkers of oxidative stress such as reduced glutathione (GSH) superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione transferase (GST) and lipid peroxidation, determined as a content of lipid hydroperoxides (LOOH) and thiobarbituric acid reactive substances (TBARS). Histopathological examination of the liver was also performed. Methods: Experimental rats were divided into five experimental groups. Exp.1: was treated with CCl4 only, Exp. 2: was treated with CCl4/L-Glu, Exp. 3: was treated with CCl4/Glu/NAC. Exp. 4: was treated with CCl4/NAC, Control 5: served as the control rats. Results: These findings suggest that the CCl4 leads to oxidative stress by depleting the antioxidant enzyme activities and increasing peroxidation products. The studied biochemical parameters were altered by the introduction of CCl4, which was normalised (to one degree or another) by L-Glu, LGlu/ NAC and NAC treatment. Conclusion: The most remarkable protective effect was observed in groups of rats that were treated with L-Glu only. This conclusion was confirmed by histopathological findings which showed less severe hepatocellular necrosis, fibrosis and inflammation in CCl4/L- Glu and CCl4/L-Glu/NAC treated group, compared to the CCl4 group.

Hepatoprotective Effect of Tagetes Erecta L. Extract on Carbon Tetrachloride Induced Hepatotoxicity in Rats

Tagetes erecta L. is a widespread garden plant that is commonly known as the marigold, and it is widely used as a medicinal herb for its anti-inflammatory, analgesic, and anti-edematous properties, which are important for phytotherapeutic, dermatological and cosmetic applications. In this study, the protective effects of water extract of Tagetes erecta L. against liver damage were evaluated in carbon tetrachloride (CCl4)-induced chronic hepatotoxicity in rats. The results showed that the treatment of extract significantly lowered the CCl4-induced serum levels of hepatic enzyme markers (GOT, GPT, ALP, and total bilirubin Liver histopathology showed that extract reduced the incidence of liver lesions including hepatic cells cloudy swelling, lymphocytes infiltration, hepatic necrosis, and fibrous connective tissue proliferation induced by CCl4 in rats. Therefore, the results of this study suggest that Tagetes erecta L. Extract could protect liver against the CCl4-induced oxidative damage in rats, and this hepatoprotective effect might be contributed to its modulation on detoxification enzymes and its antioxidant and free radical scavenger effects.
 Keywords: Tagetes erecta L., Carbon Tetrachloride, Liver Damage, Hepato-protection

Hepatoprotective properties from the seaweed Bryothamnion triquetrum (S.G.Gmelin) M.A.Howe against CCl4-induced oxidative damage in rats

Context: Seaweeds are seen as a traditional food and folk medicine by different coastal countries. The red seaweed Bryothamnion triquetrum is a widely distributed species that grows in shallow waters, and different authors have demonstrated a possible application of the seaweeds as a source of natural antioxidants and relative diseases. Aims: To evaluate the hepatoprotective properties on CCl4-induced oxidative stress in rats that were associated with the antioxidant activity from the polyphenol-rich fractions of the red seaweed Bryothamnion triquetrum. Methods: Polyphenols were determined by Folin-Cioacalteu. Antioxidant activity from phenolic compounds-rich fractions was measured by different assays (DPPH, Reducing power, β-Carotene/linoleic acid assay and Inhibition of lipoperoxidation). Aqueous extract from B. triquetrum was administered during 20 days to rats and submitted CCl4-Induced oxidative damage. The peroxidation and hepatic damage (TBARS, ASAT and ALAT), antioxidant metabolite and enzymes (glutathione, catalase and superoxide dismutase) were evaluated. Also, it was evaluated the expression of antioxidant enzymes by RT-PCR. Results: The antioxidant activity determined by different assays with polyphenolic fractions. Free Phenolic Acid was more active: DPPH, 20 µg 87%; Reducing power OD = 0.490, 20 µg ; β-carotene/linoleic acid 1 µg 53%, and inhibition of lipid peroxidation 0.250 µg 100%. Rats treated displayed lower liver TBARS, ASAT and ALAT than CCl4-treated group and catalase activity was increased. It was demonstrated expression of catalase. Conclusions: Data suggest that Bryothamnion triquetrum protects the liver against oxidative stress by modulating its antioxidant enzymes and oxidative status with potential use as phytodrug or functional food.

Potential antioxidant properties and hepatoprotective effects of Juniperus phoenicea berries against CCl4 induced hepatic damage in rats.

To investigate the antioxidant and hepatoprotective properties of Juniperus phoenicea (J.phoenicea) berries against CCl4-induced oxidative damage in rats. Hepatotoxicity was induced in albino Wistar rats by single dose of CCl4 dissolved in olive oil (1mL/kg BW, 1/1 in olive oil, i.p.). Aqueous extract of J.phoenicea berries (AEJP) was administered at the dose of 250mg/kg/day by gavage for 12 days. Obtained results revealed that administration of CCl4 caused a significant increase in plasma ASAT, ALAT, ALP and LDH activities and total bilirubin concentration, compared to the control group. While, albumin and total protein concentration were significantly lower. Additionally, a significant decrease in the level of hepatic GSH, GPx and GST activities associated with a significant increase of MDA content in CCl4 group than those of the control. However, the treatment of experimental rats with AEJP prevented these alterations and maintained the antioxidant status. The histopathological observations supported the biochemical evidences of hepatoprotection. The results of the present investigation indicate that J.Phoenicea possesses hepatoprotective activity and this effect was may be due to its antioxidant properties.

Hepatoprotective Effects of Arbutin against Liver Damage Induced by Carbon Tetrachloride in Rats

Background: The antioxidant and anti-inflammatory effects of arbutin protect against a number of diseases. Objectives: The present study evaluated the protective effect of arbutin against carbon tetrachloride (CCl4)-induced hepatotoxicity in rats. Methods: Sixty-three Wistar rats were divided into nine groups. Groups I and II were the normal control groups. Group III, the hepatotoxic group, was given CCl4. Groups IV, VI, and VIII received different dosages of arbutin along with CCl4. Groups V, VII, and IX were administered different dosages of arbutin. The albumin content, total protein, and bilirubin were assayed to determine their serum and antioxidant levels; lipid peroxidation was assessed in the serum and liver tissue. Histological studies were carried out to confirm the biochemical results. Results: Treatment with CCl4 for 28 d decreased the levels of total protein and albumin and increased the level of bilirubin and lipid peroxidation. Arbutin treatment raised the level of albumin and lowered the lipid peroxidation to normal levels. Necrosis and fibrosis were observed in the liver tissue of CCl4-injected rats, and the administration of arbutin had a protective effect on the liver tissue. Conclusions: The results of this study showed that arbutin may protect the liver against CCl4-induced oxidative damage in rats. This hepatoprotective effect might be correlated with the antioxidant and free radical scavenger effects of arbutin.

Protective Effect of Chlorogenic Acid against Carbon Tetrachloride-induced Acute Liver Damage in Rats

To evaluate the protective effect of chlorogenic acid (CGA) on carbon tetrachloride (CCl4)-induced liver injury of rats. The anti-oxidative activity of CGA was investigated with several established in vitro systems. The hepatoprotective activity of CGA against CCl4-induced acute liver injury in rats was studied. The levels of alanine aminotranferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and total bilirubin (TB) were measured. The histopathological examination was carried out to supplement the biochemical results. CGA possessed strong anti-oxidative ability in vitro. The CCl4-induced liver toxicity experiment showed that the rats pretreated with CGA (300 or 500 mg/kg) had lower levels of ALT, AST, ALP, and TB than those of the CCl4-treated group. These data were supplemented with histopathological examination of rat liver sections. CGA did not show any mortality at the dose up to 5000 mg/kg. CGA could protect the liver against CCl4-induced oxidative damage in rats, and the possible mechanism of the activity may be due to its free radical-scavenging and anti-oxidative activity.

English

The aim of the study was to investigate the protective effects of Launaea procumbens methanolic extract (LM) against CCl4-induced molecular, hormonal and pathological abnormalities in rats. Male Sprague Dawley rats were provided by National Institute of Health (NIH) Islamabad and orally fed with 100, 200 mg/kg body weight of LM after 48 h of CCl4 treatment (3 ml/kg body weight, 30% in olive oil) biweekly for 4 weeks. The results showed that the administration of LM significantly improved the CCl4-induced serum level of hormones, argyrophilic nucleolar organizer regions (AgNORs) and DNA damages. Histopathology showed that LM reduced the incidence of adrenal lesions induced by CCl4 in rats. These results suggest that LM could protect adrenal against the CCl4-induced oxidative damage in rats. Key words: Carbon tetrachloride, Launaea procumbens, adrenal histopathology.

English

Barrisal is an herbal preparation of Hamdard Laboratories (Waqf) Pakistan that has been used as hepatoprotective agent. In this study, the protective effects of Barrisal against liver damage were evaluated in carbon tetrachloride (CCl4)-induced chronic hepatotoxicity in rats. Four groups (I, II, III, IV) of Sprague-Dawley (SD) rats were used and Groups I and II were orally fed with saline and Barrisal (10 ml/kg), respectively for 14 consecutive days while Groups III and IV were orally fed with saline, Barrisal (10 ml/kg) and then a single dose of CCl4(0.3 ml/kg body weight in a 20% olive oil) was injected intraperitoneally 30 min after the last dose of Barrisal and the animals were starved for 24 h. The degree of protection was measured using biochemical parameters like serum glutamate oxaloacetate transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT), alkaline phosphatase (ALP), total protein and albumin. Data obtained from results were compared using student’s t-test (paired and unpaired) and one-way analysis of variance (ANOVA), significant differences were determined (Tukey honestly significant difference (HSD) test) using the Minitab 13 computing program. Values of P<0.05, were considered to be significant. The results showed that the treatment with Barrisal significantly lowered the CCl4-induced serum levels of hepatic enzyme markers (GOT, GPT, ALP, and total bilirubin, total cholesterol, total protein) indicated hepatoprotective effect of the Barrisal. Histopathological examination of liver sections confirmed that, pre-treatment with Barrisal reduced the hepatic damage induced by CCl4. Barrisal reduced the incidence of liver lesions including hepatic cells cloudy swelling, lymphocytes infiltration, hepatic necrosis, and fibrous connective tissue proliferation induced by CCl4 in rats. Therefore, the results of this study suggest that Barrisal could protect liver against CCl4-induced oxidative damage in rats, and this hepatoprotective effect might be because of its antioxidant and free radical scavenger effects. Key words: Barrisal, hepatoprotective, carbon tetrachloride (CCl4), hepatic enzymes marker, liver, kidney.

Carbon tetrachloride-induced lipid peroxidation and hyperglycemia in rat

Launaea procumbens methanol extract (LPME) was evaluated against carbon tetrachloride (CCl4)-induced pancreatic oxidative damage and hyperglycemia in rats. Various doses of the extract were administered to rats after 48 h of CCl4 treatment (3 ml/kg bodyweight (bw); intraperitoneally, 20% CCl4/olive oil) twice a week for 4 weeks. Coadministration of various concentrations of LPME (100, 150 and 200 mg/kg) ameliorated the toxicity of CCl4 and reversed the serum level of enzymes (amylase and lipase), glucose and hormone (insulin). The extract was able to reduce thiobarbituric acid reactive substance but increased the glutathione contents in pancreatic tissue. Depletion of antioxidant enzyme activities (catalase, peroxidase, superoxide dismutase, glutathione peroxidase, glutathione-S-transferase and glutathione-S-reductase) and DNA damages induced with CCl4 were restored by LPME supplement. It is suggested that LPME effectively protects the liver against the CCl4-induced oxidative damage in rats, possibly through antioxidant and/or free radical scavenging effects of flavonoids and phenolic compounds in the extract.

Hepatoprotective effects of Juglans regia extract against CCl4-induced oxidative damage in rats

Context: Different parts of the walnut [Juglans regia L. (Juglandaceae)] have been used in folk medicine for protection against liver injury, although its actual efficacy remains uncertain.Objective: The present study investigated the protective effect of walnut leaf extract against carbon tetrachloride (CCl4)-induced liver damage in rats.Materials and methods: The rats were randomly divided into seven groups: control, CCl4 (i.p., 0.5 mL/kg b.w., 50% CCl4 in olive oil), walnut extract (at dose level of 0.2 g/kg b.w.) alone, walnut extract (at dose levels of 0.05, 0.1, 0.2 and 0.4 g/kg b.w.) with CCl4, and treatment was carried out accordingly. On the 28th day, rats were sacrificed and blood was withdrawn by cardiac puncture. Liver damage was assessed by serum biochemical parameters (alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and albumin), antioxidant enzymes (superoxide dismutase and catalase) and histopathological observation.Results: Administration of walnut leaf extract (ranging from 0.2 to 0.4 g/kg b.w.) significantly lowered serum alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase levels in CCl4-treated rats. Walnut leaf extract increased antioxidant enzymes, including superoxide dismutase and catalase. Histopathological examination of livers showed that walnut leaves extract reduced fatty degeneration, cytoplasmic vacuolization and necrosis in CCl4-treated rats.Discussion and conclusion: These results suggest that walnut extract has a protective effect over CCl4-induced oxidative damage in rat liver. These results demonstrate that walnut extract acts as a good hepatoprotective and antioxidant agent in attenuating hepatocellular damage.

Hepatoprotective and cytoprotective properties of Hyptis suaveolens against oxidative stress–induced damage by CCl4 and H2O2

ObjectiveTo investigate capacity of Hyptis suaveolens (H. suaveolens) methanol extract as an antioxidant to protect against carbon tetrachloride (CCl4)-induced oxidative stress, hepatotoxicity in Albino Wistar rats and cytoprotective effect of hydrogen peroxide (H2O2) induced cell death in HepG2 cell line. MethodsTwo different doses of methanol extract of H. suaveolens were evaluated for the hepatoprotective activity against carbon tetrachloride (CCl4) induced hepatotoxicity in rats. Animals in Group I: served as control, group II: H. suaveolens (100 mL/kg b.w), group III: H. suaveolens (50 mL/kg b.w) + CCl4 (1 mg/kg), group IV: H. suaveolens (100 mL/kg b.w) + CCl4 (1 mL/kg) and group V: CCl4 (1 mL/kg). Histopathologic changes of liver were also evaluated. Cytotoxicity was also determined by 3, (4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT) assay. ResultsOral sigle dose treatment of CCl4 produced a marked elevation in the serum levels of aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP) and Lactate dehydrogenase (LDH). Histopathological analysis of the liver of CCl4-induced rats revealed marked liver cell necrosis with inflammatory collections that were conformed to increase in the levels of SOD, GSH, GST, GR and LPO. Treatment with H2O2 significantly induced death of HepG2 cell. Pretreatment with H. suaveolens methanol extract inhibited or attenuated H2O2 induced cytotoxicity. ConclusionsThis study shows that H. suaveolens methanol extract can be proposed to protect the liver against CCl4-induced oxidative damage in rats and protect the cells against H2O2-induced oxidative damage in HepG2 cells. The hepatoprotective and cytoprotective effects might be correlated with its antioxidant and free radical scavenger effects.

Hepatoprotective activity of Sonchus asper against carbon tetrachloride-induced injuries in male rats: a randomized controlled trial.

BackgroundSonchus asper (SAME) is used as a folk medicine in hepatic disorders. In this study, the hepatoprotective effects of the methanol extract of SAME was evaluated against carbon tetrachloride (CCl4)-induced liver injuries in rats.MethodsTo evaluate the hepatoprotective effects of SAME, 36 male Sprague–Dawley rats were equally divided into 6 groups. Rats of Group I (control) were given free access to approved feed and water. Rats of Group II were injected intraperitoneally with CCl4 (3 ml/kg) as a 30% solution in olive oil (v/v) twice a week for 4 weeks. Animals of Groups III (100 mg/kg) and IV (200 mg/kg) received SAME, whereas those of Group V were given silymarin via gavage (100 mg/kg) after 48 h of CCl4 treatment. Group VI received SAME (200 mg/kg) twice a week for 4 weeks without CCl4 treatment. Various parameters, such as the serum enzyme levels, serum biochemical marker levels, antioxidant enzyme activities, and liver histopathology were used to estimate the hepatoprotective efficacy of SAME.ResultsThe administration of SAME and silymarin significantly lowered the CCl4-induced serum levels of hepatic marker enzymes (aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase), cholesterol, low-density lipoprotein, and triglycerides while elevating high-density lipoprotein levels. The hepatic contents of glutathione and activities of catalase, superoxide dismutase, glutathione peroxidase, glutathione S-transferase, and glutathione reductase were reduced. The levels of thiobarbituric acid-reactive substances that were increased by CCl4 were brought back to control levels by the administration of SAME and silymarin. Liver histopathology showed that SAME reduced the incidence of hepatic lesions induced by CCl4 in rats.ConclusionSAME may protect the liver against CCl4-induced oxidative damage in rats.

The Protective Effects of Aqueous Extracts of Wild-Growing and Fermented Royal Sun Mushroom, Agaricus brasiliensis S. Wasser et al. (Higher Basidiomycetes), in CCl4-Induced Oxidative Damage in Rats

Culinary-medicinal Royal Sun mushroom, Agaricus brasiliensis (AbS), has traditionally been used for the prevention of a range of diseases, including cancer, hepatitis, atherosclerosis, hypercholesterolemia, diabetes, and dermatitis. The hepatoprotective effect of the fermented mushroom of A. brasiliensis (FMAE) and wild-growing A. brasiliensis (WMAE) were studied in this paper. An in vivo study of carbon tetrachloride (CCl4)-induced antioxidant activity in 2-month-old rats was conducted by examining the levels of activities of alanine aminotransaminase (ALT) and aspartate aminotransaminase (AST) and the antioxidant enzymes, including glutathione peroxidase (GSHPx) and catalase (CAT). Rats were divided into four groups, each containing six rats. The first group served as a control group. The second group was the CCl4 group. Group I and group II were treated orally with distilled water for 14 days respectively. Group III and Group IV were treated orally by WMAE and FMAE at oral doses of 50 mg/kg-day, respectively. Both WMAE and FMAE could reduce CCl4-induced toxicity, particularly hepatotoxicity, by suppressing ALT and AST activities, and increasing antioxidant enzyme activity. The studies demonstrate that both the fermented and wild-growing A. brasiliensis could protect the liver against CCl4-induced oxidative damage in rats.

Hepatoprotective Action of Radix Paeoniae Rubra Aqueous Extract against CCl4-Induced Hepatic Damage

In the present study the capacity of Radix Paeoniae Rubra aqueous extract (RPRAE) as an antioxidant to protect against carbon tetrachloride (CCl4)-induced oxidative stress and hepatotoxicity in Wistar rats was investigated. Six groups of rats were used. Radix Paeoniae Rubra aqueous extract (100 or 200 or 300 mg/kg of bw) or bifendate (100 mg/kg of bw) were given daily by gavage to the animals on 28 consecutive days to elucidate the protective effects against CCl4-induced hepatotoxicity. The 20% CCl4/olive oil was gavage of gastric tube twice a week (on the third and seventh days of each week). The animals of normal control group were given only vehicle. The animals of CCl4-treated group were administered with CCl4 twice a week (on the third and seventh days of each week) and with vehicle on rest of the days. The test materials were found effective as hepatoprotective agents, as evidenced by plasma and liver biochemical parameters. Therefore, the results of this study show that Radix Paeoniae Rubra aqueous extract can protect the liver against CCl4-induced oxidative damage in rats, and the hepatoprotective effects might be correlated with its antioxidant and free radical scavenger effects.

Hepatoprotective and Antioxidant Effects of Licorice Extract against CCl4-Induced Oxidative Damage in Rats

Licorice has been used in Chinese folk medicine for the treatment of various disorders. Licorice has the biological capabilities of detoxication, antioxidation, and antiinfection. In this study, we evaluated the antihepatotoxic effect of licorice aqueous extract (LE) on the carbon tetrachloride (CCl4)-induced liver injury in a rat model. Hepatic damage, as reveled by histology and the increased activities of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) activities, and decreased levels of serum total protein (TP), albumin (Alb) and globulin (G) were induced in rats by an administration of CCl4 at 3 mL/kg b.w. (1:1 in groundnut oil). Licorice extract significantly inhibited the elevated AST, ALP and ALT activities and the decreased TP, Alb and G levels caused by CCl4 intoxication. It also enhanced liver super oxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR), Glutathione S-transferase (GST) activities and glutathione (GSH) level, reduced malondialdehyde (MDA) level. Licorice extract still markedly reverses the increased liver hydroxyproline and serum TNF-α levels induced by CCl4 intoxication. The data of this study support a chemopreventive potential of licorice extract against liver oxidative injury.

Hepatoprotective effect of ghrelin on carbon tetrachloride-induced acute liver injury in rats

Background & AimsRecent studies have revealed that ghrelin may be an antioxidant and antiinflammatory agent. Oxidative stress are considered to play a prominent causative role in the development of various hepatic disorders. We investigated whether ghrelin plays a protective role against carbon tetrachloride (CCl4)-induced acute liver injury in rats. MethodsForty adult male Sprague–Dawley rats were randomly divided into four equal groups as; control, ghrelin, CCl4 and ghrelin plus CCl4. Evaluations were made for lipid peroxidation, enzyme activities and biochemical parameters. Pathological histology was also performed. ResultsCCl4 treatment increased plasma and liver tissue malondialdehyde (MDA) content and plasma nitric oxide (NO) level, and decreased erythrocyte and liver tissue superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities when compared to control group. At the same time, CCl4 treatment increased the serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alcaline phosphatase (ALP) activities. By contrast, ghrelin pretreatment reduced plasma and liver MDA content and plasma NO level, and increased erythrocyte and liver tissue SOD, CAT and GPx activities when compared with CCl4-treated group. Moreover, both ghrelin alone and ghrelin plus CCl4 treatment elevated serum glucose level. The CCl4-induced histopathological changes were also reduced by the ghrelin pretreatment. ConclusionOur results show that ghrelin can be proposed to protect the liver against CCl4-induced oxidative damage in rats, and the hepatoprotective effect may be correlated with its antioxidant and free radical scavenger effects.

Ameliorative effects of pycnogenol® on carbon tetrachloride‐induced hepatic oxidative damage in rats

This study evaluated the putative antioxidant activity of Pycnogenol (PYC) against CCl4-induced hepatic oxidative damage in Sprague-Dawley rats. A single oral dose of CCl4 (1.25 mL/kg) produced significantly increased levels of serum aminotransferase (AST) and alanine aminotransferase (ALT) activities. In addition, increased malondialdehyde (MDA) concentration, reduced glutathione (GSH) content, and decreased catalase, superoxide dismutase (SOD) and glutathione-S-transferase (GST) activities were observed in the hepatic tissues. However, concomitant administration with PYC (10 or 20 mg/kg) significantly improved CCl4-induced hepatic injury, as evidenced by the decline of serum AST and ALT activities in a dose dependent manner. Moreover, PYC reduced MDA concentration and increased GSH levels and catalase, SOD and GST activities in hepatic tissues, indicating that concomitant administration with PYC efficiently prevent the CCl4-induced oxidative damage in rats. The free radical scavenging assay showed that PYC has a dose-dependent scavenging activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals. These results indicate that PYC has an antioxidant effect against CCl4-induced hepatic oxidative damage and is useful as a hepatoprotective agent against various liver diseases induced by oxidative stress.