Chris A. Pritsos

Cellular distribution, metabolism and regulation of the xanthine oxidoreductase enzyme system.

Xanthine oxidase (EC 1.1.3.22) and xanthine dehydrogenase (EC 1.1.1. 204) are both members of the molybdenum hydroxylase flavoprotein family and represent different forms of the same gene product. The two enzyme forms and their reactions are often referred to as xanthine oxidoreductase (XOR) activity. Physiologically, XOR is known as the rate-limiting enzyme in purine catabolism but has also been shown to be able to metabolize a number of other physiological compounds. Recent studies have also demonstrated its ability to metabolize xenobiotics, including a number of anticancer compounds, to their active metabolites. During the past 10 years, evidence has mounted to support a role for XOR in the pathophysiology of inflammatory diseases and atherosclerosis as well as its previously determined role in ischemia-reperfusion injury. While significant progress has recently been made in our understanding of the physiological and biochemical nature of this enzyme system, considerable work still needs to be done. This paper will review some of the more recent work characterizing the interactions and the factors that influence the interactions of XOR with various physiological and xenobiotic compounds.

The mitochondrial uncoupler dicumarol disrupts the MTT assay

Dicumarol is routinely added to the 3-[4,4-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay to study the role of NAD(P)H:quinone oxido-reductase in drug activation and detoxification. We assessed the direct impact of dicumarol (a mitochondrial uncoupler) on the MTT assay. Mouse mammary tumor (EMT6) and Chinese hamster ovary (CHO) cells were treated with media containing either 10 or 1% fetal bovine serum and dicumarol (0-1000 microM) mimicking standard assay conditions. MTT, clonogenic, total reactive oxygen species (ROS), and oxygen consumption assays were performed. Significant increases in the apparent viability of EMT6 and CHO cells were observed with MTT assays after short time periods with maximum effects at 2 hr. Reduced serum concentrations intensified this effect. Conversely, significant decreases in viability for both cell lines occurred after longer incubations and serum withdrawal enhanced this effect in both cell lines. Clonogenic assays provided contrasting results where viability increased significantly only in EMT6 cells (not CHO) and was smaller than that reported by MTT. Furthermore, greater dicumarol toxicity was observed in clonogenic assays. Significant toxicity compared to control occurred after 4-hr treatment (vs. 12 hr MTT) and serum withdrawal also increased the toxicity of dicumarol with extended culture. ROS production in EMT6 and CHO cells increased in a concentration-dependent manner with 20-min dicumarol administration and thereafter declined. The EC(50) for dicumarol-induced oxygen consumption was 0.84 microM in CHO compared to 1.18 microM in EMT6 cells. Cell lines are differentially sensitive to the toxicity of dicumarol and cell survival data may be skewed by its inclusion, probably due to ROS production and mitochondrial uncoupling. Dicumarol is not recommended for inclusion in the MTT assay.

Nutrient adequacy during weight loss interventions: a randomized study in women comparing the dietary intake in a meal replacement group with a traditional food group

BackgroundSafe and effective weight control strategies are needed to stem the current obesity epidemic. The objective of this one-year study was to document and compare the macronutrient and micronutrient levels in the foods chosen by women following two different weight reduction interventions.MethodsNinety-six generally healthy overweight or obese women (ages 25–50 years; BMI 25–35 kg/m2) were randomized into a Traditional Food group (TFG) or a Meal Replacement Group (MRG) incorporating 1–2 meal replacement drinks or bars per day. Both groups had an energy-restricted goal of 5400 kJ/day. Dietary intake data was obtained using 3-Day Food records kept by the subjects at baseline, 6 months and one-year. For more uniform comparisons between groups, each diet intervention consisted of 18 small group sessions led by the same Registered Dietitian.ResultsWeight loss for the 73% (n = 70) completing this one-year study was not significantly different between the groups, but was significantly different (p ≤ .05) within each group with a mean (± standard deviation) weight loss of -6.1 ± 6.7 kg (TFG, n = 35) vs -5.0 ± 4.9 kg (MRG, n = 35). Both groups had macronutrient (Carbohydrate:Protein:Fat) ratios that were within the ranges recommended (50:19:31, TFG vs 55:16:29, MRG). Their reported reduced energy intake was similar (5729 ± 1424 kJ, TFG vs 5993 ± 2016 kJ, MRG). There was an improved dietary intake pattern in both groups as indicated by decreased intake of saturated fat (≤ 10%), cholesterol (<200 mg/day), and sodium (< 2400 mg/day), with increased total servings/day of fruits and vegetables (4.0 ± 2.2, TFG vs 4.6 ± 3.2, MRG). However, the TFG had a significantly lower dietary intake of several vitamins and minerals compared to the MRG and was at greater risk for inadequate intake.ConclusionIn this one-year university-based intervention, both dietitian-led groups successfully lost weight while improving overall dietary adequacy. The group incorporating fortified meal replacements tended to have a more adequate essential nutrient intake compared to the group following a more traditional food group diet. This study supports the need to incorporate fortified foods and/or dietary supplements while following an energy-restricted diet for weight loss.

The Production of Hydroxyl and Semiquinone Free Radicals During the Autoxidation of Redox Active Flavonoids

Catechols and other polyphenolic compounds have been shown to autoxidize and generate reactive oxygen species. A few examples of such compounds include epinephrine, 6-hydroxydopamine,2 and pyrogallol.3 We have previously reported that members of a class of polyphenolics, the flavonoids (Figure 1), behave in a similar manner. In particular four flavonoids, myricetin (3,5,7,3’,4’,5’-hexahydroxyflavone), quercetagetin (3,5,6,7,3’,4’-hexahydroxyflavone), delphinidin chloride (3,5,7,3’,4’,5’-hexahydroxyflavylium chloride) and quercetin 3,5,7,3’4’-pentahydroxy-flavone) induce cyanide-insensitive respiration in isolated beef heart mitochondrial preparations. Additionally, these compounds autoxidize in aqueous solutions at pH 7.5, as determined by measuring oxygen consumption and oxygen-dependent spectral changes. It was demonstrated that both the respiratory bursts and the autoxidation result in the production of superoxide (O2‒) and hydrogen peroxide (H2O2).4

PZ-51 (Ebselen) in vivo protection against Adriamycin-induced mouse cardiac and hepatic lipid peroxidation and toxicity

Adriamycin (Adr)-induced cardiotoxicity occurs most likely via an oxidative mechanism of action. Moderation of this activity may result in an improved therapeutic index for this compound. PZ-51, 2-phenyl-1,2-benzoisoselenazol-3(2H)-one, is a selenoorganic compound with thiol-dependent, peroxidase-like activity. We tested this compound alone and in combination with N-acetylcysteine (NAC) for its effect on Adr-induced in vivo toxicity in Balb/c mice. These studies demonstrated that PZ-51 protects against Adr-induced lipid peroxidation in heart and liver tissue and Adr-induced toxicity in general, as measured by total serum creatine kinase activity and body weight.

Antioxidant Enzymes of Larvae of the Cabbage Looper Moth, Trzchoplusza Ni: Subcellular Distribution and Activities of Superoxide Dismutase, Catalase and Glutathione Reductase

In the mid-fifth instar larvae of the cabbage looper moth, Trichoplusia ni, the subcellular distribution of total superoxide dismutase was as follows: 3.05 units (70.0%), 0.97 units (22.3%), and 0.33 units (7.6%) mg-1 protein in the mitochondrial, cytosolic and nuclear fractions, respectively. No superoxide dismutase activity was detected in the microsomal fraction. Catalase activity was unusually high and as follows: 283.4 units (47.3%), 150.1 units (25.1%), 142.3 units (23.8%), and 22.9 units (3.8%) mg-1 protein in the mitochondrial, cytosolic, microsomal (containing peroxisomes), and nuclear fractions. No glutathione peroxidase activity was found, but appreciable glutathione reductase activity was detected with broad subcellular distribution as follows: 3.86 units (36.1%), 3.68 units (34.0%), 2.46 units (23.0%), and 0.70 units (6.5%) mg-1 protein in the nuclear, mitochondrial, and cytosolic fractions, respectively. The unusually wide intracellular distribution of catalase in this phytophagous insect is apparently an evolutionary adaptation to the absence of glutathione peroxidase; hence, lack of a glutathione peroxidase-glutathione reductase role in alleviating stress from lipid peroxidation. Catalase working sequentially to superoxide dismutase, may nearly completely prevent the formation of the lipid peroxidizing .OH radical from all intracellular compartments by the destruction of H2O2 which together with O2- is a precursor of .OH.

Involvement of superoxide in the interaction of 2,3-dichloro-1,4-naphthoquinone with mitochondrial membranes.

Abstract 2,3-Dichloro-1,4-naphthoquinone (CNQ) was shown to inhibit beef heart mitochondrial respiration at separate and distinct sites in Complexes I and II of the respiratory chain. The I 50 values were 18 and 9 nmol/mg protein for the succinoxidase and NADH-oxidase enzyme systems, respectively. The interaction of CNQ with rat liver mitochondria causes a respiratory burst which is comprised of cyanide-sensitive and -insensitive components. The cyanide-sensitive portion of the CNQ respiratory burst was identified as uncoupling of oxidative phosphorylation. The cyanide-insensitive portion was shown to involve the generation of O 2 − , H 2 O 2 , and result in subsequent lipid peroxidation. The source of reducing equivalents for the CNQ-mediated generation of O 2 − and H 2 O 2 was shown to be respiratory substrate. Furthermore, CNQ promoted dose-dependent large-amplitude swelling of mitochondria that was osmotic in nature, cation nonspecific, non-energy linked, and oxygen dependent. The inclusion of cysteine in the suspension buffer prevented the CNQ-induced mitochondrial swelling. The role of O 2 − , H 2 O 2 , and peroxidation in the mitochondrial swelling induced by CNQ was discussed.

MODULATION OF GLUTATHIONE AND GLUTATHIONE DEPENDENT ANTIOXIDANT ENZYMES IN MOUSE HEART FOLLOWING DOXORUBICIN THERAPY

The toxicity of the antineoplastic agent doxorubicin (DOX) has been shown to be moderated by the antioxidant enzyme glutathione peroxidase. It has been reported that acute doses of DOX can cause an inhibition of glutathione peroxidase in cardiac tissue, that may render this tissue especially susceptible to further prooxidant damage. In this study, multiple DOX treatments at a therapeutic dose were assessed for their effect on the antioxidant enzyme status of cardiac and kidney tissue. DOX was administered i.p. (5 mg/kg) once a week for two weeks to male balb/c mice. The activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPOX) and glutathione reductase (GR) were measured 1, 2 and 7 days following the second DOX treatment in both heart and kidney. Levels of reduced glutathione (GSH) were also measured in cardiac tissue at these same times. Cardiac levels of GPOX and GR showed a time-dependent decrease in activity, with 10% and 12% inhibition for GPOX and GR, respectively, at 7 days post second treatment. Cardiac levels of GSH also showed a significant decrease, approximately 15%, at 7 days post second treatment. Cardiac levels of SOD and CAT as well as kidney levels of all four antioxidant enzymes were not affected by DOX treatment. These data suggest that DOX given in a therapeutic regimen, at a therapeutic dose, can cause decreases in cardiac levels of GPOX, GR and GSH that could render the heart especially susceptible to further oxidative challenge.

Basal and drug-induced antioxidant enzyme activities correlate with age-dependent doxorubicin oxidative toxicity.

Doxorubicin continues to be one of the most widely used anticancer agents in the clinic despite its dose-limiting side-effects. Many of doxorubicins dose-limiting toxicities occur due to its generation of toxic oxygen species, resulting in oxidative stress. Some clinical observations have suggested that doxorubicin may have greater toxicity in older patients. The studies presented here compare basal and doxorubicin-induced antioxidant enzyme activities in brain, heart, kidney and liver tissues of Fisher 344 rats of different ages to determine whether differences in these enzymes can account for the age-dependent differences observed in doxorubicin-induced toxicity. Three groups of animals were tested, young animals (2-months-old), adult animals (10-months-old) and old animals (18-months-old). The results of these studies show that in general young and adult animals have similar levels of antioxidant enzyme activity while the older animals have less. Only in the young animals is antioxidant enzyme activity significantly increased following doxorubicin treatment suggesting that enzyme induction occurs only in the young group of animals. Lipid peroxidation is shown to have the greatest increase in the old animals following doxorubicin treatment while the young animals have the smallest increase. The results from these studies suggest that there is an increase in doxorubicin-induced oxidative damage with age and that these differences may be due to basal and drug-induced differences in tissue antioxidant enzyme activities.

A redox cycling mechanism of action for 2,3-dichloro-1,4-naphthoquinone with mitochondrial membranes and the role of sulfhydryl groups.

The addition of 2,3-dichloro-1,4-naphthoquinone (CNQ) to substrate-depleted, GSH-supplemented rat liver mitochondria resulted in a dose-dependent depletion of reactable suflhydryl groups and a concomitant increase in mitochondrial disulfide content at a ratio of 2 thiols depleted/disulfide generated. The molar ratio of thiol depleted/CNQ added approached 20 at low CNQ concentrations and was unity at higher doses. The addition of CNQ to substrate-depleted mitochondrial suspensions resulted in O2 consumption which increased with increasing concentrations of mitochondria and was sensitive to N-ethylmaleimide (NEM) which establishes the ability of CNQ to interact with mitochondrial thiol redox centers. The CNQ-mediated large amplitude swelling of rat liver mitochondria was exacerbated by thiol oxidizing agents and depressed by disulfide reducing agents. A redox cycling mechanism between mitochondrial thiol groups, CNQ and oxygen was proposed to lower the matrix glutathione pool and make the mitochondria more susceptable to toxic oxygen radicals which induce swelling in isolated mitochondrial suspensions. In support of this mechanism, alpha-tocopherol was shown to prevent the CNQ-mediated swelling process. Beef heart mitochondrial NADH was oxidized by CNQ in a 1/1 molar ratio anaerobically and in a 3/1 molar ratio under aerobic conditions, whereas the fully reduced quinone, CNQH2, oxidized NADH aerobically but not anaerobically. Thus, CNQ is capable of interacting with NADH of the mitochondrial electron transport chain in a redox cycling fashion.