Effects of water-soluble antioxidant from spinach, NAO, on doxorubicin-induced heart injury.

Doxorubicin (DOX) produces clinically restorative responses in numerous human cancers, but its cardiotoxicity has limited its usefulness. Because reactive oxygen species may affect DOX-induced antitumor activity and cardiotoxicity, we evaluated the prophylactic effect of spinach natural antioxidant (NAO) on DOX-induced cardiotoxicity and oxidative stress in female Balb/c mice using histological, electron microscopical and biochemical parameters. Mice were treated with NAO for 7 days prior to and/or for 6 days after DOX administration. Pretreatment with NAO (cumulative dose: 130 mg/kg) did not hinder the effectiveness of DOX. Light and electron microscopy of DOX-treated heart revealed myocardial degeneration. When administered combined before and after DOX, NAO conferred the most significant cardiac protection. The effects of NAO on the lipid peroxidation product, malondialdehyde, and on H2O2/ hydroperoxides were examined on day 6 following DOX administration; levels of both were elevated in DOX-treated mice, compared to control. Pretreatment with NAO prevented these changes. Pretreatment with NAO before DOX administration decreased catalase and increased superoxide dismutase activities compared to the DOX group. Our results suggest usage of NAO in combination with DOX as a prophylactic strategy to protect heart muscle from DOX-induced cellular damage.

The effect of natural antioxidants, NAO and apocynin, on oxidative stress in the rat heart following LPS challenge.

Oxidative damage plays a key role in septic shock induced by lipopolysaccharide (LPS) which is known to enhance the formation of reactive oxygen species (ROS). In this study, biochemical parameters indicative of oxidative stress were tested in the rat heart following LPS challenge, with and without pretreatment with the antioxidants NAO (natural antioxidant) and apocynin. NAO is a natural antioxidant isolated and purified from spinach and its main components are flavonoids and coumaric acid derivatives. Treatment with LPS alone significantly (P<0.05) increased the malondialdehyde (MDA) level in heart, both in cytosolic and mitochondrial fractions by 1.5- and 2.4-fold, respectively, and in plasma (2.66 fold). In the heart homogenate, the level of hydroperoxides also increased significantly (P<0.05). In addition, LPS treatment significantly (P<0.05) increased NADPH oxidase activity in the heart microsomal fraction by approximately 10-fold compared to control. Pretreatment for 7 days with either apocynin or NAO prior to the LPS challenge significantly (P<0.05) improved rat survival, decreased MDA levels in both fractions and decreased microsomal NADPH-oxidase activity, compared to LPS alone. Catalase (CAT) activity slightly increased at 24 h post-LPS injection in LPS group and returned to the control level in the apocynin treated group. No meaningful changes were indicated for glutathione peroxidase activity among all the treatment groups. The activities of cytosolic and mitochondrial superoxide dismutase (SOD) enzymes significantly (P<0.05) increased approximately 20% in the LPS-treated group, compared to control. Apocynin significantly (P<0.05) decreased SOD level in the mitochondrial fraction with no effect on the cytosolic fraction; whereas, NAO had no important effect on SOD level in both fractions. The beneficial pretreatment effects of the antioxidants against oxidative stress in the rat heart presented in this study may suggest a potential chemopreventive effect of this compound in sepsis prevention.

Topical and oral administration of the natural water-soluble antioxidant from spinach reduces the multiplicity of papillomas in the Tg.AC mouse model.

The Tg.AC mouse carrying the v-Ha-ras structural gene is a useful model for the study of chemical carcinogens, especially those acting via non-genotoxic mechanisms. This study evaluated the efficacy of the non-toxic, water-soluble antioxidant from spinach, natural antioxidant (NAO), in reducing skin papilloma induction in female hemizygous Tg.AC mice treated dermally five times over 2.5 weeks with 2.5 microg 12-O-tetradecanoylphorbol-13-acetate (TPA). The TPA-only group was considered as a control; the other two groups received, additionally, NAO topically (2 mg) or orally (100 mg/kg), 5 days/week for 5 weeks. Papilloma counts made macroscopically during the clinical observations showed a significant decrease in multiplicity (P<0.01) in the NAO topically treated group. According to histological criteria, papilloma multiplicity were lower in both topical-NAO and oral-NAO groups, but significantly so only in the oral-NAO mice (P<0.01). The beneficial effect of NAO in the Tg.AC mouse is reported.

Effects of iodine on inducible nitric oxide synthase and cyclooxygenase-2 expression in sulfur mustard-induced skin.

In a previous study we demonstrated the protective effect of topical iodine as postexposure treatment for sulfur mustard (SM) application. The iodine treatment results in significantly reduced inflammation and necrosis and increased epidermal hyperplasia. The expression and localization of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) in paraffin-embedded skin samples from that study were evaluated in the present investigation. We compared the immunoreactivity of iNOS and COX-2 using five samples from each of the following four test sites: untreated control sites, SM-exposed sites, sites treated with iodine mixture 15 min after SM exposure, and sites treated with iodine 30 min after SM exposure. All animals were killed 2 days after irritant exposure. iNOS immunoreactivity was present only in skin sites exposed to SM without iodine treatment. The ulcerated skin was covered with a relatively thick band of exudate composed of iNOS-immunostained polymorphonuclear cells and macrophages. In untreated skin, COX-2 immunostaining was limited to the thin suprabasal epidermal layer. In SM-exposed skin, induction of COX-2 was noted in inflammatory cells located close to the site of epidermal injury. In skin sites treated with iodine 15 or 30 min after SM exposure, the regenerating hyperplastic epithelium showed moderate cytoplasmic staining localized to the epithelium overlying the basal layer. This pattern of staining was also present in the nearby dermal fibroblasts. Thus, in contrast to the skin samples exposed to SM without iodine treatment, the epidermal layer expressing immunohistochemical positivity for COX-2 was thicker and corresponded to the epidermal hyperplasia noted in samples treated with iodine. It is well documented that prostaglandins (PGs) promote epidermal proliferation, thereby contributing to the repair of injured skin. That the induction of the COX-2 shown in our study may also play a role in the healing process is indicated by the present evidence. The results suggest that nitric oxide radicals (NO*) are involved in mediating the damage induced by the SM and that iodine-related reduction in acute epidermal inflammation is associated with reduced iNOS expression.

Glutathione S-transferase pi expression in forestomach carcinogenesis process induced by gavage-administered 2,4-hexadienal in the F344 rat.

2,4-Hexadienal (2,4-Hx), an unsaturated aldehyde formed by in vivo and in vitro peroxidation of unsaturated lipid induced, in National Toxicology Program (NTP) gavage studies of F344 rats, forestomach hyperplasia in 13-week and 2-year exposures and squamous papilloma and carcinoma in 2-year studies. Hyperplasia was characterized by thickening of all layers of epithelium with particularly prominent proliferation of the basal cells. The present investigation describes the nature and potential significance of glutathione-S-transferase-Pi (GST-Pi) immunoexpression of normal forestomach epithelium, compared to that of 2,4-Hx-related basal cell hyperplasia and squamous cell papilloma and carcinoma. Paraffin-embedded forestomachs from these NTP studies were used to investigate possible correlations between the carcinogenic process and expression of GST-Pi, a physiological metabolic barrier and an inducible phase II detoxifying enzyme suggested to decrease the responsiveness of reactive oxygen species (ROS) and organic electrophilic compounds. The amount of immunopositive staining was graded on a scale of 0 (no staining) to 4 (marked staining). The simple basal epithelium of control rats showed strong immunopositivity. In cases of basal cell hyperplasia from the 13-week and 2-year studies, these cells usually expressed strong immunopositivity for GST-Pi (grade 3 to 4). In the 2-year treated animals only, occasional focal reduction (grade 0 to 2) in immunoreactivity for GST-Pi was noted. In papillomas and squamous cell carcinomas, a wide range of GST-Pi expression was observed, perhaps indicating irregularities in its induction or change in the phenotype of these cells compared to normal or hyperplastic ones. Reduced expression of GST-Pi by the foci of basal cell hyperplasia and in tumor cells may suggest changes in cellular protection from oxidative or electrophilic DNA damage; these changes may result in genetic alterations and be the precursor to clonal expansion.

Susceptibility of transgenic mice expressing human apolipoprotein E to closed head injury: the allele E3 is neuroprotective whereas E4 increases fatalities.

Apolipoprotein E, the major brain lipid-binding protein, is expressed in humans as three common isoforms (E2, E3 and E4). Previous studies revealed that the allele apolipoprotein E4 is a major genetic risk factor of Alzheimer's disease and that traumatic brain injury is associated with increased risk for developing this disease. Furthermore, it has been suggested that the effects of traumatic head injury and apolipoprotein E4 in Alzheimer's disease are synergistic. To test the hypothesis that the apolipoprotein E genotype affects susceptibility to brain injury, we subjected transgenic mice, expressing either human apolipoprotein E3 or human apolipoprotein E4 on a null mouse apolipoprotein E background and apolipoprotein E-deficient knockouts, to closed head injury and compared mortality, neurological recovery and the extent of brain damage of the survivors. More than 50% of the transgenic mice expressing human apolipoprotein E4 died following closed head injury, whereas only half as many of the transgenic mice expressing human apolipoprotein E3, and of the control and apolipoprotein E-deficient mice died during this period (P<0.02). A neurological severity score used for clinical assessment of the surviving mice up to 11 days after closed head injury revealed that the four mouse groups displayed similar severity of damage at 1h following injury. At three and 11 days post-injury, however, the neurological severity scores of the transgenic mice expressing human apolipoprotein E3 were significantly lower than those of the other three groups whose scores were similar, indicating better recovery of the transgenic mice expressing human apolipoprotein E3. Histopathological examination of the mice performed 11 days post-injury revealed, consistent with the above neurological results, that the size of the damaged brain area of the transgenic mice expressing human apolipoprotein E3 was smaller than that of the other head-injured groups. These findings show that transgenic mice expressing human apolipoprotein E4 are more susceptible than those expressing apolipoprotein E3 to closed head injury. We suggest that this effect is due to both a protective effect of apolipoprotein E3 and an apolipoprotein E4-related pathological function.

Effects of apocynin and natural antioxidant from spinach on inducible nitric oxide synthase and cyclooxygenase-2 induction in lipopolysaccharide-induced hepatic injury in rat.

The immunoreactivity of inducible nitric oxide synthase, and cyclooxygenase-2 was compared among groups of male Wistar rats comprising those injected with lipopolysaccharide following pretreatment with either natural antioxidant from spinach or the antioxidant apocynin, with lipopolysaccharide without pretreatment with antioxidants, with each of the two antioxidants alone, and untreated controls. The grade of staining of both inducible nitric oxide synthase and cyclooxygenase-2 increased with the severity of the inflammatory reaction in the lipopolysaccharide-treated animals, compared to the antioxidant-treated groups. Interpretation of the results of the immunostained tissues indicated that pretreatment with either antioxidant significantly (P<0.05) attenuated the lipopolysaccharide-stimulated inducible nitric oxide synthase induction. Analysis of the cycloxygenase-2-stained liver samples indicated that the pretreatment with the natural antioxidant NAO significantly (P<0.05) attenuated lipopolysaccharide-stimulated cycloxygenase-2 induction; whereas, in animals pretreated with apocynin, there was a trend of reduction in the cyclooxygenase-2 expression, but not statistically significant (P>0.05). The negative nitrotyrosine immunoreactivity of the lipopolysaccharide-related hepatic lesions may indicate that there was relatively low interaction between superoxide anions and nitric oxide to form peroxynitrite or that the expression levels of the nitrotyrosine were below the limit of detection. In all treatment groups a positive correlation (P<0.05, r=0.86) found between the inducible nitric oxide synthase and cyclooxygenase-2 scores suggests a strong relationship between these two parameters. The results indicate the possible therapeutic efficacy of NAO and apocynin in the prevention of liver damage related to clinical endotoxemia known to be associated with oxidative stress.

Increased levels of intracellular iron in the brains of ApoE-deficient mice with closed head injury.

Previous studies have revealed that apolipoprotein E (apoE)-deficient mice have distinct memory deficits and neurochemical derangements and are oxidatively stressed prior to and following closed head injury. The objective of this study was to evaluate the possibility that the enhanced susceptibility of apoE-deficient mice to closed head injury is related to impairments in their antioxidative iron-chelating mechanisms. ApoE-deficient and control mice were subjected to closed had injury, after which the extent of brain-damage and the level of iron-containing cells were assessed. Examination of the brain-damaged areas in the injured mice revealed that, by Day 3 post injury, animals of both groups were maximally and similarly affected. While the size of the damaged area of the injured control mice diminished significantly by Day 7, however recovery was not observed in injured apoE-deficient mice up to at least 14 days post-injury. Histopathologically, the decrease in the damaged areas in the control mice was interpreted as related to decreased edema. Numbers of iron-containing cells at Days 3 and 7 after injury were greater in the brains of control mice than in the apoE-deficient mice. Whereas the number of iron-containing cells in injured control mice decreased at days 9 and 14-post injury, that of the injured apoE-deficient mice plateaued by Day 9 at a level more than two-fold higher than the maximal level seen for controls. The size of the damaged areas and the number of iron-containing cells were correlated (P < 0.03) for both mouse groups at days 9 and 14 after injury. The data suggest that the increased susceptibility of apoE-deficient mice to closed head injury may be due, at least in part, to impaired iron scavenging and sustained oxidative stress.

Effects of antioxidants apocynin and the natural water-soluble antioxidant from spinach on cellular damage induced by lipopolysaccaride in the rat.

Oxidative damage plays a key role in septic shock induced by the endotoxin lipopolysaccaride (LPS) by enhancing the formation of reactive oxygen species such as superoxide anion radicals, peroxides, and their secondary product, malondialdehyde, especially in the liver. In this study, histopathologic changes in several organs were compared among groups of male Wistar rats that had been injected with LPS following prophylactic pretreatment with either of 2 antioxidants, a group that had been injected with LPS without pretreatment with antioxidants, an untreated control group, and groups that had been injected with either of the 2 antioxidants only. The antioxidants used were a water-soluble natural antioxidant from spinach (NAO) and the NADPH oxidase inhibitor apocynin. Hematoxylin-and-eosin-stained slides were prepared, and lesions were semiquantitatively scored. Exposure to LPS alone was associated with multifocal hepatocellular necrosis and acute inflammation, thymic and splenic lymphoid necrosis, ocular retinal hemorrhage and acute endophthalmitis, adrenal medullary vacuolation and necrosis and acute inflammation, and decreased adrenal cortical cytoplasmic vacuolation (consistent with depletion of steroidal hormone contents). Results indicated that pretreatment with both antioxidants for 8 days reduced, in some organs, the necrotic and inflammatory changes associated with the LPS challenge. These findings suggest a potential therapeutic application for these antioxidants in clinical sepsis.

The prophylactic effects of natural water-soluble antioxidant from spinach and apocynin in a rabbit model of lipopolysaccharide-induced endotoxemia.

Radical-scavenging antioxidants, as part of the cellular defense system, function to inhibit the formation and propagation of free radicals and active oxygen species formation. In previous studies we demonstrated that endotoxin lipopolysaccharide (LPS) promotes oxidative stress and associated pathological changes in a rat model and that use of selected antioxidants was effective in reducing LPS-related lipid peroxidation product formation in the liver, as well as LPS-related pathological changes in different organs. In this study, several toxicological parameters (ie, clinical signs, blood chemistry, and histopathological changes) were compared among groups of male New Zealand rabbits injected with LPS following prophylactic pretreatment with either of 2 antioxidants, a group injected with LPS without pretreatment with antioxidants, groups injected with either of the 2 antioxidants only, and an untreated control group. The antioxidants used were a water-soluble natural antioxidant (NAO) from spinach and the NADPH oxidase inhibitor, apocynin. Exposure to LPS alone was associated clinically with depression, tachypnea, outer ear vasodilation, and iris congestion; biochemically with a significant increase in blood total bilirubin, transaminase activity, and glucose, total cholesterol, and triglyceride levels; macroscopically with multiple whitish areas in the liver; and histologically with hepatocellular focal necrosis and acute inflammation, thymic and splenic lymphoid necrosis and depletion, acute uveitis and hemorrhages in the ciliary processes, and decreased adrenal cortical cytoplasmic vacuolation considered consistent with depletion of steroidal hormone contents. The NAO had more effective prophylactic capacities than the apocynin. The protective effects were obvious in all investigated parameters. The results indicate the possible therapeutic efficacy of NAO in the treatment of clinical endotoxemia associated with gram-negative bacterial sepsis that is known to be associated with oxidative stress.

Effect of natural antioxidants and apocynin on LPS-induced endotoxemia in rabbit.

The objective of this study was to compare the prophylactic effects of the natural antioxidant from spinach (NAO) and apocynin, on the hepatic oxidative stress and liver damage induced by lipopolysaccharide (LPS). Male New Zealand rabbits were challenged with LPS with or without 8 days of antioxidant pretreatment. Pretreatment with NAO, but not apocynin, significantly (p < 0.05) decreased the levels of hydroperoxides and malondialdehyde (MDA) in the liver cytosolic fraction and the activity of NADPH oxidase-generated superoxide in the microsomal fraction, compared to LPS alone. The activity of glutathione peroxidase (G-POX) was significantly (p < 0.05) increased in the LPS-treated group, whereas treatment with NAO, but not apocynin, significantly (p < 0.05) decreased G-POX activity. Pretreatment with the same antioxidants had no significant effects on superoxide dismutase (SOD) activity, whereas an increased level of catalase (CAT) was obtained in all LPS-treated groups. TUNEL immunohistochemical staining in the LPS-treated animals indicated that there was no increase in apoptosis outside of necrotic foci. However, apoptotic hepatocytes were observed within areas of focal necrosis in animals exposed to LPS alone or LPS plus apocynin. Hepatocyte cell proliferation was tested by the proliferating-cell nuclear antigen (PCNA) tool, which indicated a proliferative effect in the LPS group, whereas the effect disappeared in the antioxidant-treated groups. The prophylactic effect of NAO on liver pathology and the significant decreases in lipid peroxidation products and NADPH oxidase activity suggest the use of NAO as an efficient strategy for treatment of endotoxemia.

Distinct alterations in phospholipid metabolism in brains of apolipoprotein E-deficient mice.

Apolipoprotein E (ApoE) is the major brain lipoprotein and plays an important role in lipid transport. ApoE-deficient mice whose apoE gene has been knocked out have distinct cognitive and neurochemical deficits, and their recovery from brain injury is impaired. In the present study we examined the possibility that the neuronal derangements of apoE-deficient mice are related to impairments in their phospholipid metabolism. This was performed by comparison of the phospholipid, fatty acid, and cholesterol compositions of distinct membranal brain fractions of apoE-deficient and control mice. Analysis of the microsomal membrane fraction P(3) revealed that, in apoE-deficient mice, these membranes contain significantly lower levels of phosphatidylcholine (PC) than those of control mice. This effect was specific to PC and thus resulted in a twofold decrease of the PC to phosphatidylethanolamine (PE) ratio in apoE-deficient mice compared to the corresponding control ratio. In contrast, the cholesterol levels of the microsomal membranes of the two mice were similar, and the fatty acid composition of their PC was unchanged. There were, however, changes in the fatty acid composition of PE and phosphatidylserine (PS), which resulted in a lower ratio of polyunsaturated to saturated fatty acids in PE and in a higher ratio in apoE-deficient mice compared to the corresponding control values. These effects were specific to the microsomal fraction P(3) and were not observed with the brain subcellular membrane fraction P(2), which is composed mainly of plasma and mitochondrial membranes and whose phospholipid, fatty acid, and cholesterol levels were similar in apoE-deficient and control mice. These findings show that apoE deficiency results in specific and intracellular compartment-dependent changes in phospholipid metabolism, which may play an important role in mediating the neuronal effects of apoE.

Antioxidant mechanisms in apolipoprotein E deficient mice prior to and following closed head injury.

Apolipoprotein E deficient mice have distinct memory deficits and neurochemical derangements and their recovery from closed head injury is impaired. In the present study, we examined the possibility that the neuronal derangements of apolipoprotein E deficient mice are associated with oxidative stress, which in turn affects their ability to recover from close head injury. It was found that brain phospholipid levels in apolipoprotein E deficient mice are lower than those of the controls (55+/-15% of control, P<0. 01), that the cholesterol levels of the two mice groups are similar and that the levels of conjugated dienes of the apolipoprotein E deficient mice are higher than those of control mice (132+/-15% of P<0.01). Brains of apolipoprotein E deficient mice had higher Mn-superoxide dismutase (134+/-7%), catalase (122+/-8%) and glutathione reductase (167+/-7%) activities than control (P<0.01), whereas glutathione peroxidase activity and the levels of reduced glutathione and ascorbic acid were similar in the two mouse groups. Closed head injury increased catalase and glutathione peroxidase activities in both mouse groups, whereas glutathione reductase increased only in control mice. The superoxide dismutase activity was unaffected in both groups. These findings suggest that the antioxidative metabolism of apolipoprotein E deficient mice is altered both prior to and following head injury and that antioxidative mechanisms may play a role in mediating the neuronal maintenance and repair derangements of the apolipoprotein E deficient mice.

Motor and cognitive deficits in apolipoprotein E-deficient mice after closed head injury.

Previous studies suggest that traumatic brain injury is associated with increased risk factor for developing Alzheimer's disease. Furthermore, the extent of the risk seems to be most pronounced in Alzheimer's disease patients who carry the epsilon4 allele of apolipoprotein E, suggesting a connection between susceptibility to head trauma and the apolipoprotein E genotype. Apolipoprotein E-deficient mice provide a useful model for investigating the role of this lipoprotein in neuronal maintenance and repair. In the present study apolipoprotein E-deficient mice and a closed head injury experimental paradigm were used to examine the role of apolipoprotein E in brain susceptibility to head trauma and in neuronal repair. Apolipoprotein E-deficient mice were assessed up to 40 days after closed head injury for neurological and cognitive functions, as well as for histopathological changes in the hippocampus. A neurological severity score used for clinical assessment revealed more severe motor and behavioural deficits in the apolipoprotein E-deficient mice than in the controls, the impairment persisting for at least 40 days after injury. Performance in the Morris water maze, which tests spatial memory, showed a marked learning deficit of the apolipoprotein E-deficient mice when compared with injured controls, which was apparent for at least 40 days. At this time, histopathological examination revealed overt neuronal cell death bilaterally in the hippocampus of the injured apolipoprotein E-deficient mice. The finding that apolipoprotein E-deficient mice exhibit an impaired ability to recover from closed head injury suggests that apolipoprotein E plays an important role in neuronal repair following injury and highlights the applicability of this mouse model to the study of the cellular and molecular mechanisms involved.

Reduced levels of antioxidants in brains of apolipoprotein E-deficient mice following closed head injury.

Recent animal model studies using apolipoprotein E (apoE)-deficient (knockout) mice revealed that these mice have memory deficits and neurochemical derangements and that they recover from closed head injury less adequately than control mice. In the present study, we examined the possibility that the diminished recovery of apoE-deficient mice from head injury is related to a reduction in their ability to counteract oxidative damage. Measurements of reducing agents by cyclic voltammetry revealed that cortical homogenates of apoE-deficient and control mice contain similar levels of these compounds whose oxidation potentials for the two groups of mice are at 400 +/- 40 mV and 900 +/- 50 mV. The responses of the apoE-deficient and control groups to closed head injury were both biphasic and were composed of initial reductions followed by subsequent increases in the levels of reducing antioxidant equivalents. However, the two groups differed markedly in the magnitude of their response. This difference was most pronounced with the 400-mV reducing compounds, such that at 4 h after injury their levels in injured control mice increased over twofold relative to the noninjured control mice, whereas the corresponding anodic current of the apoE-deficient mice recovered only to its original level and did not increase further even by 24 h after injury. In vitro studies using recombinant apoE allele E3 and beta very low density lipoprotein revealed that this lipoprotein can delay Cu(2+)-induced lipid peroxidation. This suggests that the inability of the apoE-deficient mice to respond to brain injury by a surge in brain reducing compounds may be related, at least in part to direct antioxidant activity of apoE.

In vitro and in vivo effects of beta-carotene on rat epidermal lipoxygenases.

The in vitro and in vivo interaction between beta-carotene (BC) and lipoxygenase (LOX) was studied in rat skin. Significant in vitro inhibitory effects of BC on epidermal LOX activity were observed with both linoleic acid or arachidonic acid as substrate. Lineweaver-Burk plots for the inhibition of epidermal purified LOX indicated mixed competitive/non-competitive inhibition. In vivo effects of BC were examined in an ultraviolet A (UVA) irradiation model. Following UVA irradiation (200 Kjoule/m2) significant increases in LOX activity and malondialdehyde (MDA) values were found, whereas catalase activity was significantly decreased. Topical pretreatment of skin with BC prevented increases in LOX activity and MDA values 4 hr post-irradiation. Catalase activity was not affected by BC treatment. BC was more effective at preventing UVA induced lipid peroxidation at low then at high concentrations. Our present results indicate the protective potential of BC on in vivo UVA induced skin damage by reduction of non-enzymatic and enzymatic lipid peroxidation.

Interaction between beta-carotene and lipoxygenase in human skin.

beta-Carotene is widely used in skin care therapy. Its effects on skin are unclear, but actions on lipid peroxidation pathways may be an important element of any protection activities it exerts. This study examines the possible effects of Beta-carotene on enzymatic lipid peroxidation by lipoxygenase in human skin, using in vitro and ex vivo models. The effect of Beta-carotene on lipid peroxidation in human skin were studied in skin homogenates and in a semi-in vivo model of skin penetration, using [1-14C]-arachidonic acid or [1-14C]-linoleic acid as substrate. When relatively low concentrations (about 0.3 microM) of beta-carotene were added to epidermal homogenates, the major metabolites of arachidonic acid (12-hydroxy-cis-5,8,14, trans-10-eicosatetraenoic acid and 15-hydroxy-cis-5,8,11, trans-13-eicosatetraenoic acid) and of linoleic acid (13-hydroxy-cis-9, trans-11-octadeca dienoic acid and 9-hydroxy-trans-10, cis-12-octadeca dienoic acid) were significantly decreased. Following [1-14C]-linoleic acid penetration through the semi in vivo model layers, the skin surface was the main site in which the major linoleate product, 13-hydroxy-cis-9, trans-11-octadeca dienoic acid was detected. Furthermore, its level was inhibited by up to 80%, compared with the control, when beta-carotene was added to the system. The data presented in this study suggest possible interactions between beta-carotene and human epidermal lipoxygenase. Beta-carotene may effect lipid peroxidation in human skin, either as a free radical scavenger or as a specific lipoxygenase inhibitor.

Lipoxygenase activity in rat dermis and epidermis: partial purification and characterization.

Lipoxygenase (LOX) activity in epidermis and dermis was distributed among microsomal and cytosolic fractions. The main products of polyunsaturated fatty acid metabolism were 12-hydroperoxy-cis-5,8,14, trans-10-eicosatetraenoic acid (12-HPETE), 15-hydroperoxy-cis-5,8,11, trans-13-eicosatetraenoic acid (15-HPETE) and 13-hydroxy-cis-9, trans-11-octadecadienoic acid (13-HOD). Enzyme activities were isolated from rat dermis and epidermis by ammonium sulphate precipitation, hydrophobic chromatography and gel filtration. In the dermis, activity was found at a molecular mass of 68 kDa, a pI of 4.6 and a Km of 50 microM. This activity was inhibited by known LOX inhibitors. The main reaction products indicated that this was 15-LOX. In the epidermis, activity was found in a fraction with a molecular mass of 68 kDa, a pI of 4.6 and a Km of 80 microM. Activity was inhibited by known LOX inhibitors whereas the reaction products indicated that this was 12-LOX. LOX activity in rat skin may involve one enzyme with dual regional specificities or may comprise two different enzymes.

The interaction between beta-carotene and lipoxygenase in plant and animal systems.

The effect of beta-carotene (BC) on the activity of lipoxygenase (LOX) from plant and animal sources has been examined. Soybean lipoxygenase L-2 activity towards linoleate was inhibited by BC by a maximum of 70% at pH 6.5, whereas L-1 activity was little affected at pH 9.0. Lineweaver-Burk plots indicated that BC inhibited LOX activity by mixed competitive/non-competitive mechanisms. Other hydrophobic compounds also inhibited LOX activity; oleic acid and retinol were competitive inhibitors whereas tocopherol acetate and 5,8,11,14-eicosatetraynoic acid (ETYA) were non-competitive inhibitors. Binding studies with L-2 LOX bound to Sepharose indicated BC-binding and inhibition with the immobilized LOX. Activity of LOX from animal sources was also inhibited by BC both towards linoleate and arachidonate.

The effect of dietary vitamin E and beta-carotene on oxidation processes in the rat testis.

The effects of dietary vitamin E and beta-carotene were studied on enzymes involved in arachidonic acid metabolism and other related enzymes in the rat testis. Groups of rats were fed various soybean oil-based semi purified diets. Group 1 was fed a vitamin E-supplemented diet (+E - beta); Group 2 was fed a beta-carotene-supplemented diet (-E + beta); Group 3, the control group (-E - beta) was fed a vitamin E-deficient diet; and Group 4, the standard diet group (S), was fed vitamin E plus beta-carotene-standard diet. Soybean oxidized oil was added to the three diet groups - (+E - beta), (- E + beta) and (- E - beta), whereas the diet of S group contained non-oxidized oil. After 8 weeks rats were killed, blood and testis samples were collected for biochemical determinations. Vitamin E deficiency caused significant increase in testis thiobarbituric acid value and activities of testis NADPH oxidase, testis 15-lipoxygenase and in plasma pyruvate kinase. In contrast, significant decreases were observed in activity of testis prostaglandin synthetase, compared with antioxidant-supplemented diet groups. We also found a significant increase in 15-lipoxygenase activity in (- E + beta) diet group, compared with (- E - beta) diet group. Fatty acid analysis of testis parenchyma indicated decrease in palmitate (16:0) and arachidonate (20:4(n - 6)), and increase in oleate (18:1(n-6)) linoleate (18:2(n - 6)) and linolenate (18:3(n - 3)), when compared (-E - beta) diet group with vitamin E-supplemented diet groups. The results suggest that dietary vitamin E has a role in both enzymatic and non-enzymatic peroxidation of polyunsaturated fatty acids in the testis.