Superoxide radical formation and associated biochemical alterations in the plasma membrane of brain, heart, and liver during the lifetime of the rat.

Plasma membrane samples from rat brain, heart, and liver were examined for biochemical changes with age. A rise in superoxide radical (SOR) levels was followed by increases in thiobarbituric acid reactive substances and decreases in membrane fluidity with age. The earliest rise in SOR formation appeared in the plasma membrane from the brain. With age, protein synthesis also decreased significantly in tissue homogenates from brain and heart but was unchanged in the liver. Exposure of plasma membrane samples to in vitro-elevated SOR levels stimulated formation of lipid peroxides, as indicated by the thiobarbituric acid test, and resulted in a decrease in membrane fluidity in each tissue and in a decline in protein synthesis in brain and heart. Changes in brain lipid peroxidation and in membrane fluidity in brain and heart as a result of SOR supplementation were further enhanced due to age. In addition, the mechanism of SOR formation was examined in plasma membrane samples from the brain. SOR generation was Ca(2+)-sensitive, blocked by superoxide dismutase or vitamin E and inhibited by both indomethacin, a cyclooxygenase inhibitor, and bromophenacyl bromide, a phospholipase A2 inhibitor. These results show significant increases in SOR formation and biochemical alterations in plasma membranes from brain, heart, and liver in aging rats. SOR formation appears to be enzyme-mediated and elevated levels of this oxygen radical could be involved in membrane breakdown in older rats.     

Stimulation of phospholipase A2 by xanthine oxidase in the rat corpus luteum.

The ability of the superoxide radical (SOR) generated by xanthine oxidase to activate phospholipase A2 (PLA2) was examined in microsomes prepared from luteinized rat ovaries. Treatment of microsomes with xanthine oxidase resulted in a rapid burst in SOR formation followed by an increase in PLA2 activity. Stimulation of PLA2 activity was dose related and similar in microsomes prepared from control or prostaglandin F2 alpha (PGF2 alpha)-treated rats. Activation was inhibited by the antioxidants, vitamin E and nordihydroguaiaretic acid, and by superoxide dismutase and catalase, which metabolize SOR and H2O2 to remove reactive oxygen species from the cell. The stimulation of PLA2 activity by xanthine oxidase was dependent upon the addition of calcium ions, and it was highest in samples in which cytosol was added to membranes. These results indicate that the SOR and/or H2O2 may mediate PLA2 activation, which may be involved in the luteolytic process.     

Follow-up study of lipid peroxides, superoxide dismutase and glutathione peroxidase in the synovial membrane, serum and liver of young and old mice with collagen-induced arthritis

Because reactive oxygen species (ROS) are generally believed to play an important role in tissue injury in rheumatoid arthritis, we examined the levels of lipid peroxides, superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) in the synovial membrane, serum and liver of young (8 wk) and old (12 mo) mice with collagen-induced arthritis. In the synovial membrane, serum and liver, lipid peroxide levels of both young and old mice were increased beginning on the 3rd day after the onset of arthritis. SOD activity, which scavenges O2- and inhibits lipid peroxidation, rose markedly in the synovial membrane of young mice in parallel with the increase in lipid peroxide levels, but not so markedly in old mice. Liver GSH-Px activity, which metabolizes already formed lipid peroxides, also rose in young arthritic mice to a greater degree than in old mice. This study suggests that in inflammatory synovial lesions, lipid peroxides are generated due to an increase in ROS concentration, with resultant cytotoxicity, and that younger animals or humans can prevent this unfavorable reaction more effectively than aged ones by enzyme induction. The hypothesis that lipid peroxides formed in the oxidative lesions of the primary organ are released into the serum, trapped by the liver and metabolized there is further supported by the present study.     

Superoxide radical production in plasma membrane samples from regressing rat corpora lutea

Plasma membrane samples prepared from regressing rat corpora lutea were examined for production of the superoxide radical. A procedure was developed to purify membrane samples that were enriched approximately 15-fold with the plasma membrane marker enzyme, and superoxide radical levels were determined using electron spin resonance to measure Tiron semiquinone. During prostaglandin F2 alpha-induced and spontaneous regression, there was a significant increase in formation of superoxide radical that was not observed in plasma membrane samples from nonregressing corpora lutea. Plasma membrane incubation experiments indicated that the increase in production was temperature sensitive and reduced with inhibitors of phospholipase A2 and cyclooxygenase. Addition of superoxide dismutase or vitamin E abolished superoxide radical formation in vitro. Following the rise in superoxide radical levels during regression, there was also a significant decrease in the activity of the plasma membrane enzyme, Na+-K+ ATPase. These results indicate that the production of superoxide radical increases in plasma membrane samples prepared from regressing rat corpora lutea and that this increase is mainly due to the products of phospholipase A2 and cyclooxygenase activity.     

Changes observed in antioxidant system in the blood of postmenopausal women with breast cancer.

From experimental studies and epidemiological data, it can be inferred that lipid peroxidation is increased in cancer patients. Cases of post-menopausal, untreated women with benign and malignant breast tumours, were compared with their age matched controls in their serum lipid peroxides, antioxidant vitamins (E and C), serum selenium and serum ceruloplasmin. Erythrocyte and its membrane lipid peroxidation and antioxidant enzymes (catalase, superoxide dismutase, glutathione peroxidase and glutathione-S-transferase) levels were also analyzed. Significant increase in circulating lipid peroxides, ceruloplasmin and significant decrease in antioxidant vitamins and selenium were observed in breast cancer women. The erythrocyte and its membrane lipid peroxidation was increased significantly and severe impairment of antioxidant potential was observed in breast cancer women.     

Reversal of changes of lipid peroxide, xanthine oxidase and superoxide dismutase by cardio-protective drugs in isoproterenol induced myocardial necrosis in rats

In myocardial necrosis produced by isoproterenol (beta-adrenergic agonist) marked increase in creatine phosphokinase, phospholipase and significant decrease in cardiac glycogen and phospholipid levels were observed. The enhanced levels of lipid peroxides, xanthine oxidase activity and lowering of superoxide dismutase may lead to excessive formation of free radicals resulting in cardiac cell damage. Nifedipine--a calcium antagonist, Propranolol--a beta-blocker and guggulsterone a lipid lowering agent showed marked reversal of these metabolic changes related to ischemia induced by isoproterenol.     

Effect of Indigofera tinctoria Linn on liver antioxidant defense system during D-galactosamine/endotoxin-induced acute hepatitis in rodents

Effects of pre-treatment with the alcoholic extract of I. tinctoria (500 mg/kg body wt/day, p.o. for 21 days) on liver antioxidant defense system during acute hepatitis induced by D-galactosamine (D-GalN)/endotoxin (LPS extracted by phenol water method from E. coli serotype 0111.B4; 300 mg and 30 micrograms/kg body wt/day, i.p., 18 hr before the assay) were investigated on the activities of enzymic antioxidants such as superoxide dismutase, catalase, glutathione peroxidase and glutathione-s-transferase, and levels of total reduced glutathione in the liver of normal and experimental groups of male albino rats. Since lipid peroxidation and associated membrane damage is a key feature of D-galN/LPS-induced liver injury, the levels of lipid peroxides, was estimated and used as an index of oxidative stress. D-GalN/endotoxin-induced hepatic damage was manifested by a significant decrease in the activities of antioxidant enzymes, decreased glutathione levels and increased levels of lipid peroxides. I. tinctoria pre-treated rats showed considerable protection against D-galN/endotoxin, induced oxidative stress as evidenced by a significant increase in the activities of all the antioxidant enzymes studied and significant decrease in the levels of lipid peroxides. Results indicate that pretreatment with I. tinctoria extract in rats is very effective in reducing D-GalN/endotoxin-induced oxidative stress suggesting an antioxidant effect.     

Dynamic changes in reactive oxygen species and antioxidant levels in retinas in experimental glaucoma

We investigated the balance of free radicals in retinas at various time points after chronically elevating intraocular pressures in rats. The left eyes of female Wistar rats were divided into five intraocular pressure elevation (IOPE) subgroups after cauterization of three episcleral veins (1 day, 3 days, 1 week, 3 weeks, and 5 weeks) and time-related sham controls. Chemiluminescence levels were examined in isolated retinas. The nitroblue tetrazolium (NBT) incubation method was also performed to confirm the superoxide expression. The activities of antioxidant enzymes and lipid peroxides in isolated retinas were detected by spectrophotometry. In the IOPE group, the intraocular pressure after cauterization was around 22-30 mm Hg; chemiluminescence levels of isolated retinas were significantly elevated on day 3 and week 1; there were more NBT-positive retinal ganglion cells on day 1 and day 3; lipid peroxidation also increased significantly from day 1 and came back to the baseline on week 5; the activities of superoxide dismutases and catalase rose on week 1. Intraocular pressure elevation was shown to change the free radical balance. We suggest that free radicals and their oxidative stresses may play a role in the early stage of glaucoma in causing retinal ganglion cell death in our rat model.     

Phytotoxicity of cadmium ions on germinating seedlings of mung bean (Phaseolus vulgaris): Involvement of lipid peroxides in chlorphyll degradation

Germinating seedlings of mung bean ( Phaseolus vulgaris L. cv. K-16) were treated with different concentrations of cadmium acetate (10, 50 and 100 μ M ). Cd²⁺ lowered the chlorophyll and heme levels. The level of lipid peroxides were higher on day 3 than on day 6. However, Cd²⁺ treatment significantly enhanced the level of lipid peroxides. Similarly, a dose-dependent induction of lipoxygenase (EC 1.13.11.12) activity was observed with Cd²⁺ treatment. Further, the activities of antioxidant enzymes such as superoxide dismutase (EC 1.15.1.1) and catalase (EC 1.11.1.6) were decreased. Our results suggest that lipoxygenase-mediated accumulation of lipid peroxides on the one hand and inhibition of free radical scavenging enzymes like superoxide dismutase and catalase on the other caused a pronounced reduction in the chlorophyll and heme levels of the seedlings. The experiments conducted on the effect of Cd²⁺ on dark-grown seedlings did not conform with the result of light-grown seedlings. Though chlorophyll and heme levels decreased in a dose-dependent manner, no accumulation of lipid peroxides was observed, suggesting that the inhibition of chlorophyll synthesis by Cd²⁺ is achieved both by reaction with constituent biosynthetic enzymes as well as peroxide-mediated degradation.     

Role of antibiotics in lipid peroxidation.

Alterations in the levels of lipid peroxides, superoxide dismutase, catalase, glutathione, free fatty acid and serum ceruloplasmin were studied in rats fed with high fat cholesterol diet administered different antibiotics, viz. ampicillin, tetracycline, streptomycin, chloramphenicol and cephalosporin. The concentrations of lipid peroxides, glutathione, free fatty acid decreased in most of the tissues, except in tetracycline, streptomycin and cephalosporin treated rats. The changes observed in the activities of superoxide dismutase and catalase in the liver and kidney of these antibiotics administered groups also are in accordance with the changes in lipid peroxides. The results show that the tetracycline is hepatotoxic and nephrotoxic, while cephalosporin and streptomycin are nephrotoxic.     

Protective effect of arabic gum against cardiotoxicity induced by doxorubicin in mice: a possible mechanism of protection

Arabic gum (AG) is a naturally occurring compound that has been proposed to possess potent antioxidant activity. In this study, the possible effects whereby AG could protect against cardiotoxicity induced by doxorubicin (DOX) in mice were carried out. Administration of single dose of DOX (15 mg/kg, i.p.) induced cardiotoxicity 72 h, manifested biochemically by a significant elevation of serum creatine kinase (CK) (EC 2.7.3.2). In addition, cardiotoxicity was further confirmed by the significant increase in lipid peroxides measured as malondialdehyde (MDA). Administration of AG (25 g/kg) orally for 5 days before and 72 h after DOX injection produced a significant protection against cardiotoxicity induced by DOX. This was evidenced by significant reductions in serum CK and cardiac lipid peroxides. The effect of AG was examined on the superoxide anion radical generated by enzymatic and nonenzymatic methods. The results indicate that AG is a potent superoxide scavenger. The superoxide scavenging effect of AG may explain, at least in part, the protective effect of AG against cardiotoxicity induced by DOX.     

Folic acid supplementation inhibits NADPH oxidase-mediated superoxide anion production in the kidney

Hyperhomocysteinemia, a condition of elevated blood homocysteine (Hcy) levels, is a metabolic disease. It is a common clinical finding in patients with chronic kidney diseases and occurs almost uniformly in patients with end-stage renal disease. Hyperhomocysteinemia is also a risk factor for cardiovascular disease. Our recent studies indicate that hyperhomocysteinemia can lead to renal injury by inducing oxidative stress. Oxidative stress is one of the important mechanisms contributing to Hcy-induced tissue injury. Folic acid supplementation is regarded as a promising approach for prevention and treatment of cardiovascular disease associated with hyperhomocysteinemia due to its Hcy-lowering effect. However, its effect on the kidney is not clear. The aim of this study was to examine the effect of folic acid supplementation on Hcy-induced superoxide anion production via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in the kidney during hyperhomocysteinemia. Hyperhomocysteinemia was induced in male Sprague-Dawley rats fed a high-methionine diet for 12 wk with or without folic acid supplementation. A group of rats fed a regular diet was used as control. There was a significant increase in levels of superoxide anions and lipid peroxides in kidneys isolated from hyperhomocysteinemic rats. Activation of NADPH oxidase was responsible for hyperhomocysteinemia-induced oxidative stress in the kidney. Folic acid supplementation effectively antagonized hyperhomocysteinemia-induced oxidative stress via its Hcy-lowering and Hcy-independent effect. In vitro study also showed that 5-methyltetrahydrofolate, an active form of folate, effectively reduced Hcy-induced superoxide anion production via NADPH oxidase. Xanthine oxidase activity was increased and superoxide dismutase (SOD) activity was decreased in the kidney of hyperhomocysteinemic rats, which might also contribute to an elevation of superoxide anion level in the kidney. Folic acid supplementation attenuated xanthine oxidase activity and restored SOD activity in the kidney of hyperhomocysteinemic rats. These results suggest that folic acid supplementation may offer renal protective effect against oxidative stress.     

Effects of beta-carotene,vitamin C and E on antioxidant status in hyperlipidemic smokers

Smoking can accelerate the consumption of the stored antioxidant vitamins and increase the oxidative stress in the hyperlipidemic patients. The study investigated the effects of combined beta-carotene, vitamin C, and vitamin E on plasma antioxidant levels, erythrocyte antioxidative enzyme activities, and LDL lipid peroxides. Male hyperlipidemic smokers (35-78 years old) were randomly divided into two antioxidant supplemented groups: intervention 1 (I1, n = 22) (15 mg beta-carotene/day, 500 mg vitamin C/day, and 400 mg alpha-tocopherol equivalent/day) and intervention 2 (I2, n = 20) (30 mg beta-carotene/day, 1000 mg vitamin C/day, and 800 mg alpha-tocopherol equivalent/day). After 6-week supplementation, plasma beta-carotene, vitamin C, vitamin E, and erythrocyte glutathione levels increased significantly by 200%, 98%, 129%, and 39%, respectively, in the I1 group, and by 209%, 216%, 197%, and 32%, respectively, in the I2 group. Plasma Fe(+2) concentrations and Fe(+2)/Fe(+3) decreased significantly in both groups. Except erythrocyte glutathione peroxidase activity in the I1 group, erythrocyte catalase, glutathione peroxidase, and superoxide dismutase activities increased significantly in both groups. Lipid peroxides in LDL decreased significantly by 56% and 72% in the I1 and I2 groups, respectively. However, the levels of plasma iron, erythrocyte glutathione, and LDL lipid peroxides, and the activities of erythrocyte antioxidative enzymes did not differ between two groups. In conclusion, combined antioxidant supplements increased plasma antioxidant levels and antioxidative enzyme activities, and lowered LDL lipid peroxides in male hyperlipidemic smokers. Higher dosage of the supplements did not have an additive effect.     

Elevated serum beta-glucuronidase reflects hepatic lysosomal fragility following toxic liver injury in rats.

The level of serum beta-glucuronidase increases in various pathological conditions, including liver disorders. The aim of this investigation was to study the changes in liver lysosomal membrane stability during experimentally induced hepatic fibrosis that may result in the elevation of serum beta-glucuronidase. Liver injury was induced by intraperitoneal injections of N-nitrosodimethylamine (NDMA) in adult male albino rats over 3 weeks. The progression of fibrosis was evaluated histopathologically as well as by monitoring liver collagen content. Lipid peroxides and beta-glucuronidase levels were measured in the liver homogenate and subcellular fractions on days 0, 7, 14, and 21 after the start of NDMA administration. Serum beta-glucuronidase levels were also determined. A significant increase was observed in beta-glucuronidase levels in the serum, liver homogenate, and subcellular fractions, but not in the nuclear fraction on days 7, 14, and 21 after the start of NDMA administration. Lipid peroxides also increased in the liver homogenate and the lysosomal fraction. The measurement of lysosomal membrane stability revealed a maximum lysosomal fragility on day 21 during NDMA-induced fibrosis. In vitro studies showed that NDMA has no significant effect on liver lysosomal membrane permeability. The results of this investigation demonstrated that lysosomal fragility increases during NDMA-induced hepatic fibrosis, which could be attributed to increased lipid peroxidation of lysosomal membrane. In this study, we also elucidated the mechanism of increased beta-glucuronidase and other lysosomal glycohydrolases in the serum during hepatic fibrosis.     

Veratric acid ameliorates hyperlipidemia and oxidative stress in Wistar rats fed an atherogenic diet

An investigation was made to reveal the protective effects of veratric acid (VA), a phenolic acid against atherogenic diet-induced hyperlipidemic rats. Male albino Wistar rats were fed with atherogenic diet (4% cholesterol, 1% cholic acid, and 0.5% 2-thiouracil) daily for 30 days and treated with VA (40 mg/kg body weight) daily for a period of 30 days. Rats fed with atherogenic diet showed significant (P < 0.05) elevation in the level of plasma lipids, systolic and diastolic blood pressure, oxidative stress markers (thiobarbituric acid reactive substances, lipid peroxides) and significant (P < 0.05) reduction in the activities of enzymatic (superoxide dismutase, catalase, glutathione peroxidase) and non-enzymatic (vitamin C, vitamin E, and reduced glutathione) antioxidants in erythrocytes, plasma, and tissues (liver, kidney, and aorta). Oral administration of VA (40 mg/kg body weight) for 30 days to atherogenic diet fed rats markedly attenuates systolic, diastolic blood pressure and lipid peroxidation products. Further, VA treatment significantly improved enzymatic and non-enzymatic antioxidants levels and showed beneficial effects on lipid profile in atherogenic diet rats. All the above alterations were supported by histopathological observations. These results indicate that oral administration of VA ameliorates atherogenic diet-induced hyperlipidemia in rats by its free radical scavenging; improving the antioxidants and lipid lowering properties.     

Free radical generation, lipid peroxidation and essential fatty acids in uncontrolled essential hypertension

Vascular endothelium produces prostacyclin (PG12) and endothelium-derived vascular relaxing factor (EDRF), which are potent vasodilators and hence, may have a role in the regulation of blood pressure. Both PG12 and EDRF are readily degraded by free radicals, especially superoxide anion. Hence, we studied free radical generation and lipid peroxidation in patients with uncontrolled essential hypertension. It was observed that superoxide anion and hydrogen peroxide production by polymorphonuclear leukocytes (PMN) and the levels of lipid peroxides (measured by thiobarbituric acid assay) were higher in uncontrolled hypertensives compared to controls. Both free radical generation and the levels of lipid peroxides reverted to normal values when assayed after the control of hypertension. The calcium antagonist, verapamil, and beta-1 blocker, metoprolol, at the doses used inhibited free radical generation by phorbolmyristate acetate-stimulated PMNs. On the other hand, angiotensin II augmented free radical generation in normal PMN. In addition, it was also observed that both linoleic acid and arachidonic acid levels are low in the plasma of patients with hypertension compared to controls. These results suggest that increase in free radical generation by PMN and alterations in the plasma concentrations of essential fatty acids are closely associated with uncontrolled hypertension.     

Effect of aloe vera (Aloe barbadensis Miller) gel on doxorubicin-induced myocardial oxidative stress and calcium overload in albino rats

Administration of a single dose of doxorubicin (DOX) (7.5 mg/kg, i.v.) produces cardiotoxicity, manifested biochemically by significant decrease in blood glutathione (GSH) and tissue GSH along with elevated levels of serum lactate dehydrogenase (LDH) and serum creatine phosphokinase (CPK). In addition, cardiotoxicity was further confirmed by significant increase in lipid peroxides expressed as malondialdehyde (MDA, secondary indicator of lipid peroxidation), tissue catalase and tissue superoxide dismutase (SOD). Administration ofA. vera gel (100 and 200 mg/kg) orally for 10 days produced a significant protection against cardiotoxicity induced by DOX evidenced by significant reductions in serum LDH, serum CPK, cardiac lipid peroxides, tissue catalase and tissue SOD along with increased levels of blood and tissue GSH. The results revealed that A. vera gel produced a dose dependent protection against DOX induced cardiotoxiaty.     

镉诱导萝卜幼苗活性氧产生、脂质过氧化和抗氧化酶活性的变化

通过水培试验,研究Cd2+胁迫对萝卜幼苗活性氧的产生、脂质过氧化和抗氧化酶活性的影响。超氧 阴离子(O 2)的产生速率和丙二醛(MDA)的含量与对照相比有不同程度的增加,表明Cd2+胁迫能导致萝卜体 内的氧化胁迫;超氧化物歧化酶(SOD)的活性,随着Cd2+浓度提高,首先明显上升,然后逐渐下降,甚至低于 对照,叶片过氧化氢酶(CAT)的活性明显增加,根系CAT活性则减少,根系以及较高浓度Cd2+处理后期叶片 谷胱甘肽还原酶(GR)的活性均显著增加。推测:胁迫初期可能主要由SOD和CAT发挥抗氧化作用;后期由 于抗坏血酸—谷胱甘肽(AsA GsH)循环途径的激活,以及还原型谷胱甘肽(GSH)和植物络合素(Phytochela tins,PCs)的合成,可能在清除活性氧或者直接鏊合Cd2+中起作用。     

铝离子对二价铁离子启动的卵磷脂脂质体脂质过氧化的影响

利用化学发光、ＴＢＡ 反应与测量共轭二烯的方法观测了Ａｌ３ ＋ 对Ｆｅ２ ＋ 启动的卵磷脂脂质体脂质过氧化的影响。实验结果显示,在生理ｐＨ 条件下,Ａｌ３ ＋ 对Ｆｅ２ ＋ 启动的脂质过氧化有增强作用,表现为缩短潜伏期和加快脂质过氧化的反应速率, Ａｌ３ ＋ 的增强作用与脂质体中原先存在的过氧化物有关。这可能是因为在脂质体存在的条件下,Ａｌ３ ＋ 加速了Ｆｅ２ ＋ 的氧化,且加速作用与脂质体中原先存在的过氧化物的含量有关;另一方面,Ａｌ３ ＋ 可以引起脂质体的聚集,表现为浊度的增加;测量脂质体上标记的脂肪酸自旋标记物５ － Ｄｏｘｙｌ ｓｔｅａｒｉｃ ａｃｉｄ 的ＥＳＲ 波谱发现： Ａｌ３ ＋ 降低了脂质体的膜脂的流动性。研究表明： Ａｌ３ ＋ 对Ｆｅ２ ＋ 启动的卵磷脂脂质体的过氧化的增强作用可能与Ａｌ３ ＋ 加速了Ｆｅ２ ＋ 的氧化和改变了脂质体的物理状态有关     

Reduction of hexachlorocyclohexane-induced oxidative stress and cytotoxicity in rat liver by Emblica officinalis gaertn

The effect of prefeeding of dehydrated E. officinalis (amla) powder at 5 and 10% levels on hexachlorocyclohexane (HCH)-induced changes in multicomponent antioxidant system and lipid peroxides in rat liver was studied. HCH induced significant elevation in hepatic malondialdehyde, conjugated dienes and hydroperoxides. The prefeeding of amla at 10% level could decrease the formation of these lipid peroxides significantly. The HCH abuse resulted in a significant reduction in hepatic glutathione S-transferase (GST), glucose-6-phosphate dehydrogenase (G-6-PDH) and superoxide dismutase (SOD) activities with an elevation in the activities of glutathione peroxidase and gamma-glutamyl transpeptidase (GGT). On the other hand, the HCH-induced impairment in hepatic catalase, G-6-PDH and SOD activities were modulated by amla at the 10% level of intake. Prefeeding of amla at 5 and 10% levels appeared to reduce the HCH-induced raise in renal GGT activity. The results show the elevation of hepatic antioxidant system and reduction of cytotoxic products as a result of prefeeding of amla, which were otherwise affected by the HCH administration.