Inflammatory diseases such as hypertension are associated with high levels of oxidative stress. Characteristic of oxidative stress is the inflammatory acute phase protein response. Oxidative stress and its accompanied inflammation can be reduced via phase 2 enzyme induction. Broccoli sprouts, a rich source of phase 2 enzyme inducers such as isothiocyanates, can be incorporated into the diet to increase phase 2 enzymes. <p> The hypothesis of this study is that, the dietary intake of dried broccoli sprouts, by inducing liver phase 2 enzymes, will decrease oxidative stress and the acute phase response in the blood of spontaneously hypertensive stroke-prone rats. <p> Spontaneously hypertensive stroke-prone rats (SHRsp) and Sprague Dawley (SD) rats were placed either on a control diet of modified AIN-93G or an experimental diet of modified AIN-93G supplemented with dried broccoli sprouts. The following parameters were examined: 1. Isothiocyanate absorption (an increased level of dithiocarbamates is reflective of ITC absorption), 2. Oxidative stress (a reduction in oxidative stress is evidenced by an increase in plasma protein thiols and blood glutathione (GSH)), 3. Acute phase proteins (a decreased APR is reflected by an increase in plasma albumin and a decrease in ceruloplasmin), 4. Activity of phase 2 enzymes (increased phase 2 enzyme induction results in higher activities of liver quinone reductase (QR), glutathione-S-transferase (GST) and glutathione reductase (GR)). <p> My experimental results demonstrated that broccoli sprout feeding results in higher protein thiol levels in female SHRsp and higher blood GSH levels in males but no acute phase protein changes were observed in either male or female SHRsp. Broccoli sprout feeding caused higher QR and lower GST activities in female SHRsp but did not affect the activities of phase 2 enzymes in male SHRsp. The activities of GST and QR were higher in SD rats than in SHRsp. Levels of dithiocarbamates were higher in the broccoli fed group than in the control fed group. <p> The results from this study do not present a clear pattern to support the hypothesis that dietary intake of broccoli sprouts by inducing phase 2 enzymes will decrease parameters of oxidative stress and the acute phase response. <p> In conclusion, there is an interactive role played by animal gender and the induction of phase 2 enzymes by dried broccoli sprouts.

Myocardial ischemia-reperfusion injury occurs following the majority of cardiac events including myocardial stenosis and heart surgeries. As reactive oxygen species are one of the major contributors to ischemia-reperfusion injury, strategies to prevent their effects may be directed towards enhancing the antioxidant capacity of cells. Polyphenols, and in a more specific category, flavonoids are strong antioxidants, while possessing other biological activities such as anti-apoptotic, anti-inflammatory, and vasodilatory effects. <p>I hypothesized that flavonoids are able to reduce ischemia-reperfusion-induced cell death through multiple mechanisms including reduction of oxidative stress and induction of cellular antioxidant enzymes. The hypothesis was tested in<i> in vitro</i> and <i> in vivo</i> phases.<p>In the first phase of the studies, rat embryonic ventricular H9c2 cells were treated with various concentrations of polyphenols with or without ascorbate for 1-3 days before induction of ischemia and reperfusion. Ischemia was induced by exposure of the cells to a non-glucose containing solution bubbled with nitrogen, and reperfusion by returning the regular medium containing the corresponding polyphenols and/or ascorbate. Cell viability measurements using the MTT assay or counting acridine orange-stained cells showed that the best protection against cell death was given by catechin (44-58 %), epigallocatechin gallate (48%), proanthocyanidins (44%), and ascorbic acid (57-92%). A low concentration (10 µM) of catechin was more effective with a long-term (2 days) incubation time (64%), while a higher concentration (50 µM) could exert benefit even after 1 h pre-treatment (98%). Quercetin, resveratrol, cyanidin, and delphinidin displayed almost no protection. <P>In the second part of the in vitro study, H9c2 cells were treated with 350 to 450 µM tert-butyl hydroperoxide for 24 h after pre-incubation with various concentrations of polyphenols with or without ascorbate for either short (1 h) or prolonged (3 days) periods. Unlike in the ischemia-reperfusion experiments, 3 days pre-treatment with polyphenols did not protect and often caused cytotoxicity. In short-term (1 h) pre-treatments, the best protection was obtained with 50 µM quercetin (95%), 50 µM epigallocatechin gallate (66%), and 100 µM catechin (28%). Pre-treatment with ascorbic acid (100 µM) with or without polyphenols did not improve cell survival except in one case where it enhanced cytoprotection by epigallocatechin gallate.<p>The second phase of the studies was performed with isolated rat hearts. Rats were fed diets containing broccoli sprouts (2%), saskatoon berries (5%), or green tea extract (0.25%) for 10 days before induction of global ischemia for 20 min and reperfusion for 2 h. Broccoli sprouts decreased cell death in ischemic-reperfused hearts as assessed by caspase-3 activity (86%) and DNA fragmentation (78 %), attenuated oxidative damage as detected by lower thiobarbituric acid reactive substances (TBARS) (116%) and preserved aconitase activity (82%). Green tea extract prevented apoptosis in hearts as detected by caspase-3 activity (85%), but did not inhibit DNA fragmentation. Berries showed lower TBARS (73%). None of the feedings significantly prevented necrosis as evaluated by the release of lactate dehydrogenase into the coronary effluents, improved coronary flow, or increased heart glutathione.<p>Green tea extract was the only intervention capable of preserving the activity of glutamate cysteine ligase (78%) and quinone reductase (147%) in hearts. The sprouts group was the only group which induced these same enzymes in liver (40 and 44 %, respectively), as it was the only intervention which elevated total liver glutathione (12%). None of the interventions changed heme oxygenase-1 protein levels. Assessment of total polyphenol content revealed that broccoli sprouts had the lowest and green tea extract had the highest amount of polyphenols among the three plant materials, suggesting that the protection exhibited by broccoli sprouts was unlikely to be due to the polyphenols. <p>In conclusion, flavonoids and flavonoid-rich foods can strengthen the cellular ability to fight against oxidative stress. A part of this effect could be due to their direct antioxidant activity, while in prolonged applications they may also activate cellular pathways to promote endogenous antioxidant defences of cells. Application of low doses of flavonoids and consumption of flavonoid-rich plants in long-term ensures their effectiveness while avoiding possible toxicity. However, plants such as broccoli sprouts may have other chemical ingredients bearing biological properties which may help cells to survive states of oxidative stress.

Recent epidemiological evidence suggests that many chronic health disorders in the developed world are associated with endogenous toxins formed from the Western diet. The Western diet, which encompasses calorie dense foods, processed foods and increased quantities of red meat, can cause intracellular oxidative stress through increased formation of reactive oxygen species(ROS) and reactive carbonyl species (RCS). A number of micronutrients have been investigated for their protective capacity in in vitro and in vivo models of oxidative stress. This thesis investigated the cytotoxic targets of Fenton-mediated ROS and RCS and the subsequent protective mechanisms of vitamins B1 (thiamin) or B6 (pyridoxal, pyridoxamine or pyridoxine) in an isolated rat hepatocyte model. The approach was to use an “accelerated cytotoxicity mechanism screening” technique (ACMS) to develop an in vitro cell system that mimicked in vivo tissue cytotoxicity. Using this technique, we investigated the protective mechanisms of vitamins B1 and/or B6 against the cytotoxic effects of two endogenous toxins associated with the Western diet: 1) RCS, as exemplified by glyoxal, a glucose/fructose autoxidation product and 2) biological ROS induced by exogenous iron. Firstly, we developed an understanding of the sequence of events contributing to glyoxal-induced oxidative stress, with a focus on protein carbonylation. Next, we determined the mechanisms by which carbonyl scavenging drugs (vitamin B6 included) protected against the intracellular targets of glyoxal-induced toxicity. Our results suggested that the agents used were cytoprotective by multiple mechanisms and glyoxal trapping was only observed when the agents were administered at concentrations equal to glyoxal. We also evaluated the protective capacity of vitamins B1 and B6 against iron-catalyzed cytotoxicity and found that hepatocytes could be rescued from protein and DNA damage when vitamins B1 or B6 were added up to one hour after treatment with iron. The vitamins also varied in their primary mechanisms of protection. Our improved understanding of Western diet-derived endogenous toxins enabled us to identify and prioritize the specific inhibitory mechanisms of vitamins B1 or B6. The ability to delay, inhibit or reverse toxicity using multi-functional B1 or B6 vitamins could prove useful as therapy to minimize oxidative stress in diet-induced chronic conditions.

Hyperhomocysteinemia, a condition of elevated blood homocysteine level, is an independent risk factor for cardiovascular diseases. Folic acid can effectively reduce blood homocysteine levels. Recent studies have shown that hyperhomocysteinemia is also associated with liver disorders. However, the underlying mechanisms remain unclear. The general objective of my study was to investigate the biochemical and molecular mechanisms of homocysteine-induced liver injury and abnormal lipid metabolism. Hyperhomocysteinemia was induced in Sprague-Dawley rats by feeding a high-methionine diet for 4 weeks. An elevation of serum aminotransferases activities (indicator for liver injury) and an increase in hepatic lipid peroxidation were observed in hyperhomocysteinemic rats. Hyperhomocysteinemia-induced superoxide anion production led to oxidative stress in the liver. Reduction of oxidative stress by inhibiting superoxide anion production ameliorated hyperhomocysteinemia-induced liver injury. A significant elevation of hepatic and serum cholesterol concentrations in hyperhomocysteinemic rats was observed, exclusively due to increased expression of HMG-CoA reductase in hepatocytes. The molecular mechanisms of homocysteine-induced adverse effects were further investigated in isolated rat hepatocytes and in human hepatoma cells (HepG2). Hcy stimulated HMG-CoA reductase expression in hepatocytes via activation of transcription factors, namely, sterol regulatory element-binding protein-2 (SREBP-2), cAMP response element binding protein (CREB) and nuclear factor Y (NF-Y). Activation of these 3 transcription factors was detected in hyperhomocysteinemic rat liver and in homocysteine-treated hepatocytes. Pretreatment of hepatocytes with inhibitors for individual transcription factors effectively attenuated Hcy-induced HMG-CoA reductase mRNA expression. Supplementation of folic acid in diet significantly reduced serum homocysteine level and effectively inhibited hyperhomocysteinemia-induced superoxide anion production, resulting in amelioration of oxidative stress-mediated liver injury in hyperhomocysteinemic rats. These results reflected a protective role of folic acid in hyperhomocysteinemia-induced liver injury. In conclusion, the present study demonstrates that (1) hyperhomocysteinemia can cause oxidative stress and liver injury; (2) homocysteine stimulates cholesterol biosynthesis in hepatocytes via transcriptional regulation of HMG-CoA reductase expression; (3) supplementation of folic acid offers a hepatoprotective effect during hyperhomocysteinemia. Oxidative stress and accumulation of cholesterol in the liver contribute to liver injury associated with hyperhomocysteinemia. The role of folic acid in maintaining good health may extend beyond the cardiovascular system to encompass hyperhomocysteinemia-associated liver disorders. / October 2007

In the Ottawa Hospital Weight Management Clinic, we have previously identified subpopulations of patients in the upper and lower quintiles for rate of weight loss, and characterized them as ‘obese diet sensitive’ (ODS) and ‘obese diet resistant’ (ODR) patient groups, respectively. Skeletal muscle is a major contributor to basal metabolic rate and mitochondrial proton leak in skeletal muscle can account for up to 50 % of resting oxygen consumption. The overall aim of this research is to explore differences in mitochondrial function in human primary myotubes from ODS and ODR subjects. Subsets of ODS and ODR subjects (n = 9/group) who followed a hypocaloric clinical weight loss program at the Ottawa Weight Management Clinic consented to a muscle (vastus lateralis) biopsy. Human primary myoblasts obtained from biopsies were immunopurified and differentiated into myotubes. Mitochondrial function and distribution were compared in intact myotubes from ODS and ODR subjects. Mitochondrial proton leak was significantly lower (p< 0.05) in ODR myotubes compared to ODS myotubes, independent of whether cells were differentiated in low or high glucose medium. In addition, in low glucose medium, ODR myotubes had higher MnSOD protein levels compared to ODS myotubes (p< 0.05). However, there were no significant differences in mitochondrial content, mitochondrial membrane potential, cellular ROS levels or ATP content between ODS and ODR myotubes. Overall, our in vitro mitochondrial proton leak results are consistent with our previous ex vivo results. Future research should examine the possibility that differences in proton leak between ODS and ODR groups may be related to mechanisms of cellular ROS regulation.

Every year, several thousand individuals suffer spinal cord injury (SCI) in North America, while 1.5 million suffer traumatic brain injury in the U.S.A. alone. Primary mechanical trauma to the CNS is followed by a complex pathology, including vascular dysregulation, ischemia, edema and traumatic hemorrhage. Secondary damage is to a large extent caused by oxidative stress and inflammatory processes, resulting in necrosis and apoptosis of neural cells. If secondary tissue injury could be limited by interference with any of the pathomechanisms involved, preservation of structure and function would increase the potential for functional recovery. Experiments performed in other laboratories have shown that the polyphenolic flavonoid quercetin acts as an anti-oxidant and anti-inflammatory, reduces edema formation and apoptotic cell death. Quercetin is also an excellent iron chelator. This action profile suggested a high therapeutic potential for acute CNS trauma. Therefore, I used models of both spinal cord injury and head trauma in adult male rats to test the hypothesis that administration of quercetin is beneficial for the therapy of acute traumatic CNS injury. While the primary focus of my work was on therapy of acute traumatic spinal cord injury, quercetin was also evaluated in the settings of chronic SCI and acute head trauma. I found that, in a rat model of mid-thoracic spinal cord compression injury, 1) administration of quercetin, starting 1 hr after injury and continued every 12 hr, improved recovery of motor function in the hind limbs in more than half of the injured animals to a degree that allowed previously paraplegic animals to step or walk. The minimum quercetin dose that was efficacious was 5 µmol/kg. The minimum treatment duration for optimal outcome was determined to be 3 days. In control animals, some spontaneous recovery of motor function did occur, but never to an extent that allowed animals to step or walk. Quercetin administration was associated with more efficient iron clearance from the site of injury, decreased inflammatory response as reflected in decrease of myeloperoxidase activity and decreased apoptosis of neural cells at the site of injury. 2) Quercetin administered in the same injury model as late as 2 weeks after injury, given in a higher dose than that used for treatment in the acute phase, still resulted in significant recovery of motor function in 40% of the injured animals, although at a lower level of performance, when compared to early onset of treatment. 3) Quercetin administered after moderate fluid percussion brain injury resulted in decreased oxidative stress, as reflected in higher tissue glutathione levels at the site of injury. In animals receiving quercetin, the amplitude of compound action potentials was significantly better maintained at 24 hr and 72 hr after injury than in saline-treated control animals. My experiments have shown that the flavonoid quercetin is neuroprotective in a rat model of brain trauma and in a rat model of spinal cord injury. My data show that administration of quercetin after CNS trauma promotes iron clearance, decreases oxidative stress and inflammation. Quercetin also decreases apoptotic cell death following neurotrauma. These results suggest that quercetin may be a valuable adjunct in the therapy of acute CNS trauma. There is a possibility that administration of quercetin may be beneficial even in certain settings of chronic CNS trauma. These conclusions are based solely on the results from animal experiments. However, the fact that few adverse reactions have been noted to date in either animal experiments or human trials targeting other diseases is encouraging for the progression to human clinical trials for patients with spinal cord injury.

Previous studies on fishes exposed to effluent from the Key Lake uranium mill in northern Saskatchewan have demonstrated elevated lipids in young-of-the-year pike (Esox lucius), deformities in larval pike and decreased survival of fathead minnows (Pimephales promelas). The objectives of this thesis were to evaluate possible factors that could be contributing to altered bioenergetics of juvenile northern pike inhabiting lakes receiving effluent from the Key Lake operation and to examine the effects of effluent exposure on biomarkers of oxidative stress and histopathology of target organs. Although glycogen and triglycerides stores were significantly greater in pike from exposure lakes compared to the reference, triglycerides stores of juvenile pike prey items showed no overall differences among lakes. Measures of parasitism, however, were negatively correlated with pike bioenergetics thereby reflecting a possible energetic cost of parasitism on reference lake fish. The degree of infection by intestinal parasites and gill monogeneans was greatest in reference pike and intermediate in low exposure pike, whereas high exposure pike harboured no parasites. <p>Arsenic, nickel and selenium are elevated in lakes downstream of the Key Lake mill and have been shown to be associated with increased reactive oxygen species (ROS) in biological systems causing oxidative stress. The potential for oxidative stress was assessed in pike liver and kidney using several biomarkers. Overall, the concentrations of total, reduced and oxidized glutathione and the ratio of oxidized to reduced glutathione did not differ significantly among exposure and reference pike. The activity of glutathione peroxidase was greater in high exposure than reference liver whereas, contrary to predictions, lipid peroxidation was greater in reference than exposure pike tissues. <p>Histopathological evaluations revealed greater kidney and gill pathology in reference lake pike, whereas for liver, hepatocyte morphology differed among lakes without any clear signs of pathology. Trace metal analyses of muscle showed that eight elements (arsenic, cobalt, copper, iron, molybdenum, selenium, thallium, uranium) were significantly elevated in exposure pike. These results provide only limited evidence of oxidative stress in exposure pike tissues and no evidence of histopathology despite indications that metals are bioaccumulating in tissue. Overall, the results from this thesis suggest that the health and condition of juvenile northern pike living downstream of the Key Lake uranium mill may not be compromised by effluent exposure.

A series of seven experiments were conducted to evaluate the risk of acute (sudden death syndrome; SDS) or chronic (congestive heart failure; CHF) heart failure associated with dietary over-supplementation of vitamin A, vitamin D3, vitamin E, vitamin C or cardiotoxic factors present in meat meal. The risk of heart failure associated with the above mentioned dietary factors was tested followed by gross, microscopic, ultrastructural and biochemical investigation for mechanisms associated with mentioned risk factors. Simultaneously, the molecular mechanisms underlying the deterioration of heart function in fast-growing commercial broilers were studied. Each compound was tested separately at a concentration higher than the recommended levels. The basic experimental unit comprised groups of 40 to 50 day old male broiler chickens at the start of experiment. Lowered thermal brooding temperature protocol, an approach resulting in clinical manifestation of heart failure in practically all broilers predisposed to heart disease, was used.<p> Broilers fed the vitamin D3 enriched diet were 2.5 fold more likely to succumb to acute heart failure (p<0.05). Simulated stress challenge with epinephrine revealed that broilers fed excess of vitamin D3 were more susceptible to ventricular arrhythmia. The risk of CHF was higher (P<0.05) in broilers fed the vitamin D3, vitamin A and methanol soluble extract from meat meal enriched diets as compared to groups fed the control diet. The incidence of CHF in broilers fed the diet fortified with vitamin E was not significantly different as compared to the control group, whereas supplementation of vitamin C in the diet tended (p=0.10) to reduce the incidence of CHF. The level of malondialdehyde equivalents, an indicator of lipid peroxidation, was significantly higher (p<0.05) in myocardium of broilers developing CHF irrespective of dietary factors. Antioxidant vitamins (E and C) did not prevent lipid peroxidation in broilers developing CHF. <p>In conclusion, the present findings indicate that over-supplementation of vitamin A and D3 increases the risk of heart failure in broilers. Meat meal contains some unknown cardiotoxic factors, capable of precipitating heart conditions in susceptible broilers. Oxidative stress is involved in the pathogenesis of heart failure in broilers, but supplementation of antioxidant vitamins did not prevent oxidative damage in broilers that developed CHF. The oversupplementation of vitamins (A and D3) should not be encouraged in broilers diet as they may increase the economic losses to broilers industry subsequent to heart related mortalities/morbidities.

Myocardial ischemia-reperfusion injury occurs following the majority of cardiac events including myocardial stenosis and heart surgeries. As reactive oxygen species are one of the major contributors to ischemia-reperfusion injury, strategies to prevent their effects may be directed towards enhancing the antioxidant capacity of cells. Polyphenols, and in a more specific category, flavonoids are strong antioxidants, while possessing other biological activities such as anti-apoptotic, anti-inflammatory, and vasodilatory effects. <p>I hypothesized that flavonoids are able to reduce ischemia-reperfusion-induced cell death through multiple mechanisms including reduction of oxidative stress and induction of cellular antioxidant enzymes. The hypothesis was tested in<i> in vitro</i> and <i> in vivo</i> phases.<p>In the first phase of the studies, rat embryonic ventricular H9c2 cells were treated with various concentrations of polyphenols with or without ascorbate for 1-3 days before induction of ischemia and reperfusion. Ischemia was induced by exposure of the cells to a non-glucose containing solution bubbled with nitrogen, and reperfusion by returning the regular medium containing the corresponding polyphenols and/or ascorbate. Cell viability measurements using the MTT assay or counting acridine orange-stained cells showed that the best protection against cell death was given by catechin (44-58 %), epigallocatechin gallate (48%), proanthocyanidins (44%), and ascorbic acid (57-92%). A low concentration (10 µM) of catechin was more effective with a long-term (2 days) incubation time (64%), while a higher concentration (50 µM) could exert benefit even after 1 h pre-treatment (98%). Quercetin, resveratrol, cyanidin, and delphinidin displayed almost no protection. <P>In the second part of the in vitro study, H9c2 cells were treated with 350 to 450 µM tert-butyl hydroperoxide for 24 h after pre-incubation with various concentrations of polyphenols with or without ascorbate for either short (1 h) or prolonged (3 days) periods. Unlike in the ischemia-reperfusion experiments, 3 days pre-treatment with polyphenols did not protect and often caused cytotoxicity. In short-term (1 h) pre-treatments, the best protection was obtained with 50 µM quercetin (95%), 50 µM epigallocatechin gallate (66%), and 100 µM catechin (28%). Pre-treatment with ascorbic acid (100 µM) with or without polyphenols did not improve cell survival except in one case where it enhanced cytoprotection by epigallocatechin gallate.<p>The second phase of the studies was performed with isolated rat hearts. Rats were fed diets containing broccoli sprouts (2%), saskatoon berries (5%), or green tea extract (0.25%) for 10 days before induction of global ischemia for 20 min and reperfusion for 2 h. Broccoli sprouts decreased cell death in ischemic-reperfused hearts as assessed by caspase-3 activity (86%) and DNA fragmentation (78 %), attenuated oxidative damage as detected by lower thiobarbituric acid reactive substances (TBARS) (116%) and preserved aconitase activity (82%). Green tea extract prevented apoptosis in hearts as detected by caspase-3 activity (85%), but did not inhibit DNA fragmentation. Berries showed lower TBARS (73%). None of the feedings significantly prevented necrosis as evaluated by the release of lactate dehydrogenase into the coronary effluents, improved coronary flow, or increased heart glutathione.<p>Green tea extract was the only intervention capable of preserving the activity of glutamate cysteine ligase (78%) and quinone reductase (147%) in hearts. The sprouts group was the only group which induced these same enzymes in liver (40 and 44 %, respectively), as it was the only intervention which elevated total liver glutathione (12%). None of the interventions changed heme oxygenase-1 protein levels. Assessment of total polyphenol content revealed that broccoli sprouts had the lowest and green tea extract had the highest amount of polyphenols among the three plant materials, suggesting that the protection exhibited by broccoli sprouts was unlikely to be due to the polyphenols. <p>In conclusion, flavonoids and flavonoid-rich foods can strengthen the cellular ability to fight against oxidative stress. A part of this effect could be due to their direct antioxidant activity, while in prolonged applications they may also activate cellular pathways to promote endogenous antioxidant defences of cells. Application of low doses of flavonoids and consumption of flavonoid-rich plants in long-term ensures their effectiveness while avoiding possible toxicity. However, plants such as broccoli sprouts may have other chemical ingredients bearing biological properties which may help cells to survive states of oxidative stress.

The pathology of multiple sclerosis (MS) is characterized by an inflammatory mononuclear infiltration in the white matter. There has been converging evidence of the oxidative stress playing a role in the onset and progression of MS. We postulated that the decreasing oxidative stress might help in the management of MS. We know that the induction of phase 2 enzymes decreases the oxidative stress. The experimental allergic encephalomyelitis (EAE) induced in the Lewis rats were used to test this hypothesis. The 24 animals were placed into two groups: 1) those on normal rat chow, 2) those on rat chow containing 7.5 g/kg of tetra-butylhydroxyanisole (BHA), a food preservative. All the animals were administered 100 µg of guinea pig myelin basic protein in their tails to induce EAE and examined daily in a double blinded fashion. On 29th day of the induction, the animals were sacrificed, blood collected for glutathione (GSH) measurements and tissues collected for histology. All the animals, regardless of their diet status, developed symptoms of EAE on different days ranging from tail weakness to hind limb paralysis and all of them reached remission of acute EAE before the 28th day of induction. The non-BHA fed animals developed hind limb weakness in 8 animals and hind limb paralysis in 4 cases, while that of BHA fed group developed tail paralysis in 2, hind limb weakness in 2 and hind limb paralysis in 8 cases. The histology of the non-BHA group correlated well with the clinical symptoms of perivascular mononuclear infiltration. However, the BHA group revealed complete pathological recovery. Animals with BHA in the diet had significantly raised GSH, indicating the induction of phase 2 enzymes. We conclude that dietary phase 2 enzyme inducers show potential therapeutic benefits in EAE and should be examined for this role in MS.