Intracellular adhesion molecule-1 up-regulation on thyrocytes by iodine of non-obese diabetic.H2(h4) mice is reactive oxygen species-dependent.

Intracellular adhesion molecule-1 (ICAM-1) expression on the thyroid follicular cells of non-obese diabetic (NOD).H2(h4) mice is enhanced by iodide treatment, which correlates with autoimmune thyroid disease in genetically susceptible NOD.H2(h4) mice. The current study examines the mechanism of iodine-enhanced up-regulation of ICAM-1 on the surface of thyroid cells. We hypothesized that the up-regulation of ICAM-1 is due to a transient increase in production of reactive oxygen species (ROS). ROS may initiate signalling of the ICAM-1 gene promoter, enhancing up-regulated ICAM-1 protein on the cell surface. Single-cell suspensions of thyroid follicular cells from thyroiditis-susceptible NOD.H2(h4) or non-susceptible BALB/c mice were treated in vitro with sodium iodide. Extracellular and intracellular ROS were assessed by luminol-derived chemiluminescence and flow cytometry assays respectively. Our results demonstrate that thyroid follicular cells of NOD.H2(h4) generate higher levels of ROS compared with cells from non-susceptible strains of mice. Expression of a subunit protein of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, p67(phox), was analysed by Western blot immunoassay. A constitutive expression of the p67(phox) subunit protein was observed in NOD.H2(h4) mice prior to iodine treatment. No such expression was found in BALB/c mice. Treatment of NOD.H2(h4) thyroid cells with diphenyleneiodium, an inhibitor of NADPH oxidase, reduced generation of ROS and of ICAM-1 protein expression. Thus, thyrocytes from NOD.H2(h4) mice produce enhanced levels of ROS that may be mediated by NADPH oxidase. Consequently, in NOD.H2(h4) mice the ROS-induced signal for ICAM-1 up-regulation may contribute to mononuclear cellular infiltration of the thyroid gland and the progression of autoimmune thyroid disease.

Age-related increase in mitochondrial superoxide generation in the testosterone-producing cells of Brown Norway rat testes: relationship to reduced steroidogenic function?

Aging in Brown Norway rats is accompanied by the reduced production of testosterone by the Leydig cells, the testicular cells responsible for synthesizing and secreting this essential steroid. As yet, the mechanism by which Leydig cell steroidogenesis is reduced is unknown. Herein we assess the production of mitochondrial reactive oxygen species by intact Leydig cells isolated from the testes of young and old rats. To this end, Leydig cells were incubated with lucigenin (bis-N-methylacridinium nitrate), a probe that enters cells, localizes to mitochondria, and yields a significant chemiluminescent response following its reaction with intramitochondrial superoxide. Leydig cells from old rats elicited significantly greater lucigenin-derived chemiluminescence (LDCL) than those from young rats. Electron microscopic stereological analysis revealed that the absolute volume of mitochondria in the old cells was reduced from that in the young. These results, taken together, suggest that there are age-related changes in the production of reactive oxygen species by the mitochondria of Leydig cells, with those of old Leydig cells producing significantly greater levels than those of young Leydig cells. The results are consistent with the proposal that mitochondrial-derived reactive oxygen may play a role in the irreversible decline in the ability of old Leydig cells to produce testosterone.

Enzyme-mediated dialdehyde formation: an alternative pathway for benzo[a]pyrene 7,8-dihydrodiol bioactivation.

Polycyclic aromatic hydrocarbons, such as benzo[a]pyrene, are widespread environmental carcinogens of human concern. Several enzymatic systems have been shown to activate benzo[a]pyrene 7, 8-dihydrodiol, the proximate carcinogenic metabolite of benzo[a]pyrene, to a reactive species which produces both a chemiluminescence response and genotoxic lesions. The chemiluminescence response has been proposed to be the result of the formation of a dioxetane which upon ring opening forms a reactive dialdehyde intermediate. In in vitro incubations involving phorbol ester-stimulated human polymorphonuclear leukocytes or an isolated enzyme system consisting of myeloperoxidase, taurine, and hydrogen peroxide, a prolonged (>60 min) chemiluminescence response was observed from benzo[a]pyrene 7,8-dihydrodiol. HPLC analysis of the reaction mixture revealed the existence of a product which is dependent upon both taurine and the hydrocarbon. Characterization of this product using UV, NMR, and MS indicated that the product is a pyrene with two side chains resulting from bond breakage of a ring, yielding a dialdehyde. These side chains contain a portion of taurine covalently attached through imine formation with the aldehydes resulting from dioxetane ring opening. Replacement of taurine with either protein or DNA also produced a prolonged chemiluminescence response. These results demonstrate for the first time the formation of a novel electrophilic species from benzo[a]pyrene 7,8-dihydrodiol which along with an increased production of photons from this activation mechanism may lead to DNA and/or protein damage that is different from that elicited by diol epoxides.

Mitochondrial adaptations to obesity-related oxidant stress.

It is not known why viable hepatocytes in fatty livers are vulnerable to necrosis, but associated mitochondrial alterations suggest that reactive oxygen species (ROS) production may be increased. Although the mechanisms for ROS-mediated lethality are not well understood, increased mitochondrial ROS generation often precedes cell death, and hence, might promote hepatocyte necrosis. The aim of this study is to determine if liver mitochondria from obese mice with fatty hepatocytes actually produce increased ROS. Secondary objectives are to identify potential mechanisms for ROS increases and to evaluate whether ROS increase uncoupling protein (UCP)-2, a mitochondrial protein that promotes ATP depletion and necrosis. Compared to mitochondria from normal livers, fatty liver mitochondria have a 50% reduction in cytochrome c content and produce superoxide anion at a greater rate. They also contain 25% more GSH and demonstrate 70% greater manganese superoxide dismutase activity and a 35% reduction in glutathione peroxidase activity. Mitochondrial generation of H(2)O(2) is increased by 200% and the activities of enzymes that detoxify H(2)O(2) in other cellular compartments are abnormal. Cytosolic glutathione peroxidase and catalase activities are 42 and 153% of control values, respectively. These changes in the production and detoxification of mitochondrial ROS are associated with a 300% increase in the mitochondrial content of UCP-2, although the content of beta-1 ATP synthase, a constitutive mitochondrial membrane protein, is unaffected. Supporting the possibility that mitochondrial ROS induce UCP-2 in fatty hepatocytes, a mitochondrial redox cycling agent that increases mitochondrial ROS production upregulates UCP-2 mRNAs in primary cultures of normal rat hepatocytes by 300%. Thus, ROS production is increased in fatty liver mitochondria. This may result from chronic apoptotic stress and provoke adaptations, including increases in UCP-2, that potentiate necrosis.

Role of quinones in toxicology.

Quinones represent a class of toxicological intermediates which can create a variety of hazardous effects in vivo, including acute cytotoxicity, immunotoxicity, and carcinogenesis. The mechanisms by which quinones cause these effects can be quite complex. Quinones are Michael acceptors, and cellular damage can occur through alkylation of crucial cellular proteins and/or DNA. Alternatively, quinones are highly redox active molecules which can redox cycle with their semiquinone radicals, leading to formation of reactive oxygen species (ROS), including superoxide, hydrogen peroxide, and ultimately the hydroxyl radical. Production of ROS can cause severe oxidative stress within cells through the formation of oxidized cellular macromolecules, including lipids, proteins, and DNA. Formation of oxidatively damaged bases such as 8-oxodeoxyguanosine has been associated with aging and carcinogenesis. Furthermore, ROS can activate a number of signaling pathways, including protein kinase C and RAS. This review explores the varied cytotoxic effects of quinones using specific examples, including quinones produced from benzene, polycyclic aromatic hydrocarbons, estrogens, and catecholamines. The evidence strongly suggests that the numerous mechanisms of quinone toxicity (i.e., alkylation vs oxidative stress) can be correlated with the known pathology of the parent compound(s).

Increased mitochondrial superoxide production in rat liver mitochondria, rat hepatocytes, and HepG2 cells following ethinyl estradiol treatment.

Ethinyl estradiol (EE) is a strong promoter of hepatocarcinogenesis. Treatment of rats with EE and other hepatic promoters induces a mitosuppressed state characterized by decreased hepatocyte turnover and reduced growth responsiveness. Previously, we identified several nuclear and mitochondrial genome-encoded mitochondrial genes whose transcripts were increased during EE-induced hepatic mitosuppression in rats and in EE-treated HepG2 cells (Chen et al. Carcinogenesis, 17, 2783-2786, 1996 and Carcinogenesis, 19, 101-107, 1998). In both cultured rat hepatocytes and HepG2 cells, EE increased respiratory chain activity (reflected by increased mitochondrial superoxide production detected as increased lucigenin-derived chemiluminescence (LDCL). In this paper, we provide additional characterizations of these effects. Increased LDCL was detected in mitochondria isolated from EE-treated rats, documenting that these estrogen effects on mitochondrial function are not confined to cells in culture. EE and estradiol (E2) increased LDCL in cultured rat hepatocytes and HepG2 cells in a dose- (beginning at 0.25 microM levels) and time-dependent response. Inhibition of P450-mediated estrogen metabolism inhibited, while direct exposure to E2 catechol metabolites enhanced LDCL. Co-treatment with glutathione ester or with the specific antiestrogen, ICI 182708 inhibited LDCL. In contrast, estrogen-induced LDCL was enhanced by glutathione depletion, and by inhibition of catechol-o-methyltransferase. These results support a working hypothesis that in liver cells, increased respiratory chain activity induced by estrogen treatment requires both metabolism to catechols and an estrogen receptor-mediated signal transduction pathway.

Biochemical characterization of lucigenin (Bis-N-methylacridinium) as a chemiluminescent probe for detecting intramitochondrial superoxide anion radical production.

Direct detection of intramitochondrial superoxide anion radical (O(-*)(2)) production is of critical importance for investigating the pathophysiological consequences resulting from altered cellular reactive oxygen homeostasis. The purpose of this study with isolated mitochondria was to characterize the biochemical basis for lucigenin as a chemiluminescent probe to detect intramitochondrial O(-*)(2) production. Incubation of isolated mitochondria with lucigenin at non-redox cycling concentration produced lucigenin-derived chemiluminescence (LDCL), which was increased markedly by mitochondrial substrates, pyruvate/malate or succinate. The LDCL was reduced greatly by the membrane permeable superoxide dismutase (SOD) mimetics, 2,2,6,6-tetramethylpiperidine-N-oxyl and Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin, but not by Cu,Zn-SOD. With an ion-pair HPLC method, a concentration-dependent accumulation of lucigenin was detected within mitochondria. The accumulation of lucigenin by mitochondria was reduced markedly in the presence of carbonyl cyanide p-(trifluoromethoxy)phenyhyldrazone, an uncoupler known to dissipate the mitochondrial membrane potential. With submitochondrial particles, we observed that both complexes I and III of the mitochondrial electron transport chain appear to be able to catalyze the one electron reduction of lucigenin, a critical step involved in LDCL. After incubation of mitochondria with lucigenin at non-redox cycling concentrations, formation of N-methylacridone, the proposed end product of the reaction pathway leading to LDCL, within the mitochondrial fraction was also detected. In addition, a significant linear correlation was observed between the LDCL and either the lucigenin accumulation or the N-methylacridone formation within the mitochondria. Taken together, our results conclusively demonstrate that when properly used LDCL can reliably detect intramitochondrial O(-*)(2) production.

Phorbol ester-induced mononuclear cell differentiation is blocked by the mitogen-activated protein kinase kinase (MEK) inhibitor PD98059.

The purpose of this study was to evaluate whether the mitogen-activated protein kinase (MAPK) signaling pathway contributes to 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mononuclear differentiation in the human myeloblastic leukemia ML-1 cells. Upon TPA treatment, the activity of ERK1 and ERK2 rapidly increased, with maximal induction between 1 and 3 h, while ERK2 protein levels remained constant. The activity of JNK1 was also significantly induced, with JNK1 protein levels increasing moderately during exposure to TPA. Treatment of cells with PD98059, a specific inhibitor of mitogen-activated protein kinase kinase (MEK), inhibited TPA-induced ERK2 activity. Furthermore, PD98059 completely blocked the TPA-induced differentiation of ML-1 cells, as assessed by a number of features associated with mononuclear differentiation including changes in morphology, nonspecific esterase activity, phagocytic ability, NADPH oxidase activity, mitochondrial respiration, and c-jun mRNA inducibility. We conclude that activation of the MEK/ERK signaling pathway is necessary for TPA-induced mononuclear cell differentiation.

Detection of mitochondria-derived reactive oxygen species production by the chemilumigenic probes lucigenin and luminol.

Both lucigenin and luminol have widely been used as chemilumigenic probes for detecting reactive oxygen species (ROS) production by various cellular systems. Our laboratory has previously demonstrated that lucigenin localizes to the mitochondria of rat alveolar macrophages and that lucigenin-derived chemiluminescence (CL) appears to reflects superoxide O2(-.) production by mitochondria in the unstimulated macrophages. In this study, we further examined the ability of lucigenin- and luminol-derived CL to assess O2(-.) and H2O2 formation, respectively, by isolated intact mitochondria. Mitochondria were isolated from monocytes/macrophages differentiated from monoblastic ML-1 cells. Incubation of the substrate-supported mitochondria with lucigenin at non-redox cycling concentration produced lucigenin-derived CL. Luminol-derived CL was also elicited with substrate-supplemented mitochondria in the presence of horseradish peroxidase (HRP). The lucigenin-derived CL was diminished extensively by the membrane permeable superoxide dismutase (SOD) mimetics, 2,2,6, 6-tetramethylpiperidine-N-oxyl and Mn(III) tetrakis(1-methyl-4-pyridyl)porphyrin, but not by Cu,Zn-SOD. On the other hand, luminol-derived CL was not observed in the absence of HRP and was significantly inhibited by catalase. A spectrum of agents known to specifically affect mitochondrial respiration exhibited corresponding effects on both lucigenin- and luminol-derived CL. Taken together, our results demonstrate that with isolated mitochondria lucigenin-derived CL monitors intramitochondrial O2(-.) production by the mitochondrial electron transport chain, whereas the luminol-derived CL detects H2O2 released from the mitochondria. As such, use of both probes provides a comprehensive and clear assessment of ROS production by mitochondria.

Phenotypic abnormalities in macrophages from leptin-deficient, obese mice.

Obesity is a complex syndrome that involves defective signaling by a number of different factors that regulate appetite and energy homeostasis. Treatment with exogenous leptin reverses hyperphagia and obesity in ob/ob mice, which have a mutation that causes leptin deficiency, proving the importance of this factor and its receptors in the obesity syndrome. Cells with leptin receptors have been identified outside of the appetite regulatory centers in the brain. Thus leptin has peripheral targets. Because macrophages express signaling-competent leptin receptors, these cells may be altered during chronic leptin deficiency. Consistent with this concept, the present study identifies several phenotypic abnormalities in macrophages from ob/ob mice, including decreased steady-state levels of uncoupling protein-2 mRNA, increased mitochondrial production of superoxide and hydrogen peroxide, constitutive activation of CCAAT enhancer binding protein (C/EBP)-beta, an oxidant-sensitive transcription factor, increased expression of interleukin-6 and cyclooxygenase (COX)-2, two C/EBP-beta target genes, and increased COX-2-dependent production of PGE2. Given the importance of macrophages in the general regulation of inflammation and immunity, these alterations in macrophage function may contribute to obesity-related pathophysiology.

Metabolism of benzo[a]pyrene and benzo[a]pyrene-7,8-diol by human cytochrome P450 1B1.

Benzo[a]pyrene (B[a]P), a ubiquitous environmental, tobacco and dietary carcinogen, has been implicated in human cancer etiology. The role of human cytochrome P450 1B1 in the metabolism of B[a]P is poorly understood. Using microsomal preparations of human P450 1A1, 1A2 and 1B1 expressed in baculovirus-infected insect cells, as well as human and rat P450 1B1 expressed in yeast, we have determined the metabolism of B[a]P, with and without the addition of exogenous epoxide hydrolase, and B[a]P-7,8-dihydrodiol (7,8-diol), each substrate at a concentration of 10 microM. HPLC analysis detected eight major metabolites of B[a]P and four metabolites of the 7,8-diol. The results of these studies indicate that cytochrome P450 1B1 carries out metabolism of B[a]P along the pathway to the postulated ultimate carcinogen, the diol epoxide 2, at rates much higher than P450 1A2 but less than P450 1A1. The rates of formation of the 7,8-diol metabolite in incubations with epoxide hydrolase are 0.17 and 0.38 nmol/min/nmol P450 for human P450 1B1 and 1A1, respectively, and undetectable for 1A2. The rates of total tetrol metabolite formation from the 7,8-diol, which are indicative of diol epoxide formation, are 0.60, 0.43 and 2.58 nmol/min/nmol P450 for 1B1, 1A2 and 1A1 respectively. In agreement with other reports of rat P450 1B1 activity, our data show this rat enzyme to be very active for B[a]P and 7,8-diol, with rates higher than human P450 1B1. In addition to the established role of P450 1A1 in B[a]P metabolism, P450 1B1 may significantly contribute to B[a]P and 7,8-diol metabolism and carcinogenesis in rodent tumor models and in humans.

Somatic mutations of the mitochondrial genome in human colorectal tumours.

Alterations of oxidative phosphorylation in tumour cells were originally believed to have a causative role in cancerous growth. More recently, mitochondria have again received attention with regards to neoplasia, largely because of their role in apoptosis and other aspects of tumour biology. The mitochondrial genome is particularly susceptible to mutations because of the high level of reactive oxygen species (ROS) generation in this organelle, coupled with a low level of DNA repair. However, no detailed analysis of mitochondrial DNA in human tumours has yet been reported. In this study, we analysed the complete mtDNA genome of ten human colorectal cancer cell lines by sequencing and found mutations in seven (70%). The majority of mutations were transitions at purines, consistent with an ROS-related derivation. The mutations were somatic, and those evaluated occurred in the primary tumour from which the cell line was derived. Most of the mutations were homoplasmic, indicating that the mutant genome was dominant at the intracellular and intercellular levels. We showed that mitochondria can rapidly become homogeneous in colorectal cancer cells using cell fusions. These findings provide the first examples of homoplasmic mutations in the mtDNA of tumour cells and have potential implications for the abnormal metabolic and apoptotic processes in cancer.

Identification of dithiolethione-inducible gene-1 as a leukotriene B4 12-hydroxydehydrogenase: implications for chemoprevention.

Cancer chemoprevention is inhibition of neoplastic disease by naturally occurring or synthetic chemical agents. Dithiolethiones inhibit production of experimentally produced tumors by elevating the expression of several genes that encode for known cytoprotective enzymes. In an effort to discover additional molecular mechanisms mediating chemoprevention, cDNA clones representing a gene that is transcriptionally activated by dithiolethiones, hence named dithiolethione-inducible gene-1 (DIG-1), were isolated from rat liver via differential hybridization screening. The deduced amino acid sequence of DIG-1 was found to have 80% identity with the human liver enzyme leukotriene B4 (LTB4) 12-hydroxydehydrogenase. DIG-1, purified >400-fold from the liver of rats dosed with 1,2-dithiole-3-dithiolethione, possessed an NADP+-dependent activity to convert LTB4 to 12-oxo-LTB4. Kinetic analysis of DIG-1 revealed apparent Km and Vmax values of 28 mM and 8.1 nmol 12-oxo-LTB4 formed/min/mg purified protein respectively. Since LTB4 is a potent chemotactic factor and stimulator of production of reactive oxygen species from neutrophils, the effects of DIG-1 on these LTB4-mediated processes were examined. Pre-incubation of LTB4 with purified rat hepatic DIG-1 greatly diminished LTB4-stimulated migration of neutrophils. In addition, pre-incubation of LTB4 with purified rat hepatic DIG-1 reduced LTB4-stimulated production of superoxide anions in neutrophils, as evidenced by decreased lucigenin-derived chemiluminescence. These results suggest that DIG-1-catalyzed dehydrogenation of LTB4 to 12-oxo-LTB4 inhibits the pro-inflammatory actions of LTB4. Consequently, elevation of LTB4 catabolism via enhanced DIG-1 activity may suppress inflammatory processes implicated in tumorigenesis.

Validation of lucigenin (bis-N-methylacridinium) as a chemilumigenic probe for detecting superoxide anion radical production by enzymatic and cellular systems.

Lucigenin is most noted for its wide use as a chemiluminescent detector of superoxide anion radical (O2-.) production by biological systems. However, its validity as a O2-.-detecting probe has recently been questioned in view of its ability to undergo redox cycling in several in vitro enzymatic systems, which produce little or no O2-.. Whether and to what extent lucigenin redox cycling occurs in systems that produce significant amounts of O2-. has not been carefully investigated. We examined and correlated three end points, including sensitive measurement of lucigenin-derived chemiluminescence (LDCL), O2 consumption by oxygen polarography, and O2-. production by 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide spin trapping to characterize the potential of lucigenin to undergo redox cycling and as such to act as an additional source of O2-. in various enzymatic and cellular systems. Marked LDCL was elicited at lucigenin concentrations ranging from 1 to 5 microM in all of the O2-.-generating systems examined, including xanthine oxidase (XO)/xanthine, lipoamide dehydrogenase/ NADH, isolated mitochondria, mitochondria in intact cells, and phagocytic NADPH oxidase. These concentrations of lucigenin were far below those that stimulated additional O2 consumption or O2-. production in the above systems. Moreover, a significant linear correlation between LDCL and superoxide dismutase-inhibitable cytochrome c reduction was observed in the XO/ xanthine and phagocytic NADPH oxidase systems. In contrast to the above O2-.-generating systems, no LDCL was observed at non-redox cycling concentrations of lucigenin in the glucose oxidase/glucose and XO/NADH systems, which do not produce a significant amount of O2-.. Thus, LDCL still appears to be a valid probe for detecting O2-. production by enzymatic and cellular sources.

Diphenyleneiodonium, an NAD(P)H oxidase inhibitor, also potently inhibits mitochondrial reactive oxygen species production.

Diphenyleneiodonium (DPI) has frequently been used to inhibit reactive oxygen species (ROS) production mediated by flavoenzymes, particularly NAD(P)H oxidase. This study was undertaken to examine if DPI could also inhibit production of superoxide and H2O2 by mitochondria, the major source of cellular ROS. Detection of mitochondrial superoxide by lucigenin-derived chemiluminescence (CL) with unstimulated monocytes/macrophages showed that DPI at concentrations that inhibit NAD(P)H oxidase markedly diminished the production of superoxide by mitochondrial respiration. Similarly, the extracellular H2O2 derived from mitochondrial respiration as detected by luminol-derived CL in the presence of horseradish peroxidase was also greatly reduced by DPI. DPI was as potent as rotenone in inhibiting the production of superoxide and H2O2 by mitochondrial respiration. With substrate-supported isolated mitochondria, DPI was shown to reduce mitochondrial superoxide production probably through inhibiting NADH-ubiquinone oxidoreductase (complex I).

Enhanced levels of several mitochondrial mRNA transcripts and mitochondrial superoxide production during ethinyl estradiol-induced hepatocarcinogenesis and after estrogen treatment of HepG2 cells.

Ethinyl estradiol (EE) is a strong hepatic promoter and weak complete hepatocarcinogen. Among the effects on rat liver caused by chronic exposure to non-hepatotoxic doses of EE is an initial, transient increase in hepatocyte growth followed by a subsequent inhibition (mitosuppression) of basal and/or induced liver growth. To investigate the mechanism of EE-induced mitosuppression, we performed a differential display and identified 10 genes whose expression was increased 2- to 4-fold in EE-induced, mitosuppressed livers (Chen et al., Carcinogenesis, 17, 2783-2786, 1996). We found that one of these clones was homologous to nuclear genome-encoded mitochondrial ATP synthase subunit E. Here, we describe the identification of two additional cDNAs representing transcripts whose levels were elevated during EE-induced mitosuppression as mitochondrial DNA-encoded cytochrome c oxidase subunit III and ATP synthase 6. In addition, we found that EE, estradiol and the estradiol catechol metabolites, 4-OH-estradiol and 2-OH-estradiol, increased the levels of these and other mitochondrial genome-encoded transcripts in human hepatoma HepG2 cells. We also observed that this increase can be blocked by inhibition of cytochrome P450-mediated estrogen metabolism, and that this increase is accompanied by increased mitochondrial superoxide production, which reflects increased respiratory chain activity.

Increased susceptibility of Alzheimer's disease temporal cortex to oxygen free radical-mediated processes.

Reactive oxygen-mediated processes are though to contribute to the pathogenesis of Alzheimer's disease (AD). To investigate this hypothesis we studied autopsy tissue from 11 pairs of AD cases and control individuals matched for age, postmortem delay, and tissue storage time. The temporal neocortex, which is severely involved by AD pathology, and the cerebellum, which is spared, were analyzed for tissue markers of lipid peroxidation (LPO). The average chemiluminescence formed from bond breakage in tissue homogenates during a 3-h incubation, without the presence of catalysts such as metal ions or ascorbate, was significantly increased in the AD temporal cortex to 130% of matched controls. Basal tissue content of LPO products (thiobarbituric acid reactive substances--TBARs) was not different between groups. However, TBARs were significantly elevated in AD temporal cortex to 135% of control after the incubation. In contrast, in the cerebellum there was no difference between AD and control tissue, indicating a disease-specific tissue effect. Because the use of oral antioxidants have received considerable attention in the last few years, the results seen in the testing of an AD patient who took daily vitamin E supplements for 4 years is particularly interesting. The time course for CL reactivity in the temporal cortex was considerably delayed compared to all other samples. This observation is consistent with the hypothesis that antioxidants within tissue will quench ROS-mediated reactions. This study indicates that there is increased susceptibility to ROS in the AD temporal cortex that may contribute to the pathogenesis of the disease. Furthermore, our observation suggest that oral antioxidant supplementation may be protective against LPO in the human brain.

Analysis of target cell susceptibility as a basis for the development of a chemoprotective strategy against benzene-induced hematotoxicities.

A goal of our research is to identify biochemical factors that underlie the susceptibility of bone marrow cell populations to benzene metabolites so as to develop a mechanistically based chemoprotective strategy that may be used in susceptible humans exposed to benzene. By doing biochemical risk analysis of bone marrow stromal cells from mice and rats and the human myeloid cell lines, HL-60 and ML-1; and by using buthionine sulfoximine and dicumarol we have observed that the susceptibility of these cell populations to hydroquinone (HQ) correlates with their concentration of glutathione (GSH) and activity of quinone reductase (QR). Accordingly, the induction of QR and GSH by 1,2-dithiole-3-thione (D3T) in these cell populations has resulted in a significant protection against the following hydroquinone-mediated toxicities: inhibition of cell proliferation and viability; reduced ability of stromal cells to support myelopoiesis; and altered differentiated of ML-1 cells to monocytes/macrophages. Preliminary in vivo experiments indicate that feeding mice D3T results in an induction of QR in the bone marrow compartment such that stromal cells are more resistant to hydroquinone-induced cytotoxicity in vitro. Overall, these studies suggest that in addition to hepatic cytochrome P4502E1, bone marrow QR and GSH are factors that could determine an individual's relative susceptibility to the toxic effects of benzene.