Elizabeth Shuler. A Thesis

Transcription

1 THE EFFECTS OF FLAVONOIDS ON MITOCHONDRIAL MEMBRANE- ASSOCIATED REDUCED PYRIDINE NUCLEOTIDE-UTILIZING SYSTEMS OF ADULT HYMENOLEPIS DIMINUTA (CESTODA) AND ASCARIS SUUM (NEMATODA) Elizabeth Shuler A Thesis Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of Master of Science August 2013 Committee: Carmen Fioravanti, Advisor Lee Meserve Vipaporn Phuntumart

2 2013 Elizabeth Shuler All Rights Reserved

3 ABSTRACT iii Carmen Fioravanti, Advisor The adult intestinal cestode, Hymenolepis diminuta, is energetically anaerobic and displays mitochondria that physiologically function anaerobically. These organelles require an inner membrane-associated NADPH NADP transhydrogenase as well as other membraneassociated, NADH-utilizing activities that reflect anaerobic electron transport. Plant flavonoids are known to affect both mammalian and invertebrate systems and, more specifically, the ecdysone 20-monooxygenase, mitochondrial transhydrogenase and electron transport-linked systems of the tobacco hornworm, Manduca sexta. However, with the exception of the isofavonoid, rotenone, no data existed as to the potential effects of plant flavonoids on parasitic helminth anaerobic, mitochondrial enzymes. Thus, the effects of chrysin, quercetin, morin, juglone, and plumbagin on the H.diminuta transhydrogenase as well as NADH dehydrogenase, NADH-cytochrome c reductase, and an NADH NAD transhydrogenation were evaluated. Although lacking an NADPH NAD system, comparisons with the other corresponding mitochondrial activities of another anaerobic, intestinal, adult helminth, viz., the nematode, Ascaris suum, were made. Activities were assessed spectrophotometrically employing isolated cestode or nematode mitochondrial membranes as the source of enzyme activities. While not all flavonoid treatments proved to significantly affect the activities tested, a suggested inhibition by chrysin was noted and stimulations by juglone and plumbagin were noted for the helminth transhydrogenase. The H. diminuta NADH dehydrogenase was inhibited by plumbagin, but stimulation was apparent at higher concentrations whereas morin stimulated (lower concentrations) and inhibited (higher concentration) and plumbagin stimulated the A. suum

4 enzyme. Both the cestode and nematode NADH-cytochrome c reductase activities appeared to iv respond positively to the presence of juglone. A tendency towards stimulation of the NADH NAD transhydrogenation of H.diminuta by plumbagin, juglone and chrysin were observed while morin was somewhat inhibitory. In contrast, the A. suum NADH NAD was inhibited by chrysin and juglone, but tended towards stimulation by plumbagin. The study presented apparently represents the first attempts to assess the effects of these plant flavonoids on key mitochondrial systems of the anaerobic, parasitic helminths. Lastly, comparisons of the helminth mitochondrial systems with the mitochondrial, NADPH-dependent ecdysone 20- monooxygenase and the NADPH NAD transhydrogenase systems of M. sexta are made.

5 ACKNOWLEDGMENTS v I would like to give a very special thanks to Dr. Carmen Fioravanti for his guidance and support over the years. His constant patience and understanding encouraged me to continue my research even when I could not see the light at the end of the tunnel. I would also like to thank my lab mate, Carl Breidenbach, for his aid and support throughout my thesis research. His knowledge on lab research was of invaluable help, as was his humor and moral support. I would like to thank my brothers, Andy and Kevin, and especially my mother, Priscilla, for their continued emotional support as well as their encouragement, especially whenever I wanted to give up my work. I would like to thank Chris Drummond for not only helping me prepare for my defense but for being a support system throughout the defense preparations. His understanding of the stress involved as well as his patience with me helped me to realize it is possible to get through the defense. I would also like to thank my committee members, Dr. Vipaporn Phuntumart and Dr. Lee Meserve. I would like to extend my thanks to Dr. Phuntumart for agreeing at the last minute to be a member of my graduate committee. I would also like to extend my thanks to Dr. Meserve for his guidance throughout all my years at Bowling Green State University. I would also like to thank him for giving me the chance to be a teaching assistance for his anatomy and physiology class, thus giving me the opportunity to discover my love for teaching anatomy and physiology.

6 I would like to thank the Department of Biological Sciences at Bowling Green State vi University for aiding in my research by funding my research. I would also like to thank Sigma Xi, The Scientific Research Society for their Grant in Aid of Research for funding my research.

7 vii TABLE OF CONTENTS Page CHAPTER I. LITERATURE REVIEW... 1 Hymenolepis diminuta... 1 Ascaris suum... 5 Flavonoids Purpose CHAPTER II. THE STUDY INTRODUCTION MATERIALS AND METHODS RESULTS Effects of flavonoids on H. diminuta Effects of flavonoids on A. suum DISCUSSION LITERATURE CITED APPENDIX A. INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC) 44 Approval Information... 44

8 viii LIST OF FIGURES/TABLES Figure/Table Page 1 Life cycle of Hymenolepis diminuta Pathway of primary carbohydrate utilization in Hymenolepis diminuta Life cycle of Ascaris suum Pathway of carbohydrate dissimilation in Ascaris muscle Effects of flavonoids on the NADPH NAD transhydrogenase of adult Hymenolepis diminuta mitochondrial membranes Effects of flavonoids on the NADH dehydrogenase of adult Hymenolepis diminuta mitochondrial membranes Effects of flavonoids on the NADH cytochrome c reductase of adult Hymenolepis diminuta mitochondrial membranes Effects of flavonoids on the NADH NAD transhydrogenation of adult Hymenolepis diminuta mitochondrial membranes Effects of flavonoids on the NADH dehydrogenase of adult Ascaris suum mitochondrial membranes Effects of flavonoids on the NADH cytochrome c reductase of adult Ascaris suum mitochondrial membranes Effects of flavonoids on the NADH NAD transhydrogenation of adult Ascaris suum mitochondrial membranes... 32

9 1 CHAPTER I: LITERATURE REVIEW Hymenolepis diminuta Hymenolepis diminuta is an intestinal cestode of mammals. It is a member of the kingdom Animalia, phylum Platyhelminthes, class Cestoda, order Cyclophyllidea, and family Hymenolepidae. It is chiefly a parasite of the rat, and thus is referred to as the rat tapeworm, although less common human infections do occur (Roberts and Janovy, 2000). The life cycle of H. diminuta is presented in Figure 1. Eggs are passed in the feces of the definitive host, i.e., the rat. Mature eggs are ingested by an arthropod intermediate host, usually the flour beetle, Tribolium confusum. Once mature eggs have been ingested by the intermediate host, the eggs hatch, and oncospheres are released. The oncospheres attach to the intestinal wall of the arthropod, penetrate the intestine, and enter the haemocoel, where they mature further, thereby developing into cysticercoids. The developed cysticercoids are the infective stage of the parasite. The definitive host becomes infected when it eats an infected intermediate host. Upon entering the definitive host, the cysticercoids excyst, allowing the infective larvae to attach to the intestinal wall by way of the four suckers on the scolex or head. With attachment, the immature worms develop into mature adult worms 21 days post-infection. The mature worm is segmented, and the segments are termed proglottids. Eggs are found in the terminal proglottids, which also contain both male and female reproductive organs. The terminal proglottids break off and are shed in the feces of the rat, thus releasing the eggs and starting the life cycle once again (Smyth, 1976).

10 2 Figure 1: Life cycle of H. diminuta. From CDC website on Hymenolepiasis (CDC 2009). (1) Eggs are passed in feces of an infected mammal. (2) Infective eggs are ingested by an arthropod intermediate host. (3) Eggs hatch, releasing oncospheres. (4) Oncospheres attach to intestinal wall of arthropod, penetrate intestine and enter haemocoel, where they continue development into mature cysticercoids. (5) Mammalian definitive host ingests infective arthropod, thus becoming infected once cysticercoids excysts and attach to intestinal wall of definitive host. (6) Larvae develop into mature worms, and eggs are shed from terminal proglottids.

11 3 The metabolism of H. diminuta is essentially energetically anaerobic, with succinate accumulating as the major end product along with lesser amounts of acetate and lactate (Scheibel and Saz, 1966; Scheibel et al., 1968). As presented in Figure 2, succinate is formed via the utilization of cytosolic malate. In adult H. diminuta, glycolysis begins with the dissimilation of glucose to phosphoenolpyruvate (PEP). In the cytosol, glucose is converted to PEP with the concomitant reduction of NADP P to NADH. PEP serves as the substrate for COR2R fixation by the enzyme PEP carboxykinase, thereby yielding oxaloacetate (OAA). OAA is reduced to malate by malate dehydrogenase with concomitant NADH oxidation to NADP P. Malate enters the mitochondria wherein it serves as the substrate for a dismutation reaction. Approximately half of the malate is acted on by the NADP-specific malic enzyme to produce pyruvate, COR2, Rand NADPH. The remaining is converted to fumarate by fumarase. In H. diminuta and other helminth parasites, the mitochondrial, inner membrane-associated NADPH NADP P transhydrogenase plays an essential role in connecting NADP-specific malate oxidation, via the malic enzyme, with the NADH-requiring electron transport-coupled fumarate reductase, thereby fostering ATP generation. This occurs by hydride ion transfer from malic enzyme- generated NADPH to NADP P, producing the required NADH. This inner membrane-associated transhydrogenase reaction thus fulfills the vital need for hydride ion transfer (Saz et al., 1972; Fioravanti and Saz, 1975; Fioravanti and Vandock, 2010).

12 4 Figure 2: Pathway of primary carbohydrate utilization in H. diminuta (Saz and Lescure, 1969; Fioravanti and Saz, 1976; Fioravanti and Saz, 1980).

13 5 Ascaris suum Another essentially anaerobic helminth that has been well studied is the nematode Ascaris suum, an intestinal roundworm of pigs. A variety of this nematode, viz., A. lumbricoides, infects humans. A. lumbricoides often can be found in high numbers in human populations that are living in conditions of poor hygiene (Smyth, 1976). More recently, molecular studies indicated that A. suum and A. lumbricoides are one and the same organism (Leles et al., 2012). This nematode is a member of the kingdom Animalia, phylum Nematoda, class Secernentea, order Ascaridida, family Ascarididae (Smyth, 1976). The life cycle of A. suum is presented in Figure 3. Adult worms live in the lumen of the small intestine of the definitive host. Eggs are passed in the feces of the infected definitive host. Before fertilized eggs can be infective, they must embryonate in the appropriate environment of warm, moist, and shaded soil for 18 days to several weeks. The larva becomes infective in the egg after the first molt (Johnstone, 2000). The conditions of the environment in which the fertilized eggs must mature before becoming infective plays a role in how long it takes for the eggs to embryonate and become infective. The optimum environmental conditions are moist, warm, shaded soil. Once the infective eggs are ingested, the larvae hatch and invade the mucosal layer of the small intestine. Here, the second molt takes place (Johnstone, 2000). The larvae are then carried to the lungs via the portal and then systemic circulation. The larvae remain in the lungs where they further mature for about ten to 14 days. After the ten to 14 day maturation period, the larvae penetrate the wall of the alveoli, ascend the bronchiole tree and enter the

14 6 trachea and then the oral cavity, where they are swallowed. Upon reaching the small intestine, the third and fourth molts occur and the larvae mature into adult worms (Johnstone, 2000).

15 7 Figure 3: Life cycle of A. suum. From CDC website on Ascariasis. (1) Eggs are passed in feces of infected host. (2) Fertilized eggs develop in soil for 18 days to several months, finally becoming infective. Unfertilized eggs will not become infective. (3) Mature fertilized eggs are ingested by host, and continue development. (4) Larvae penetrate mucosal lining of small intestine, enter liver via the hepatic portal circulatory system, and migrate to the lungs via the systemic circulatory system. (5) Larvae are coughed up, and then swallowed again, where they mature into adult worms in the small intestine.

16 8 The anaerobic mitochondrial metabolism of A. suum is similar to that of H. diminuta (Fig.4). Unlike H. diminuta, however, the mitochondria of A. suum contain a NADP P-linked malic enzyme system (Saz and Lescure, 1969; Fioravanti and Saz, 1980; Saz, 1981). Succinate, as well as fatty acids derived from succinate, are the major end products of the anaerobic metabolism of A. suum (Saz and Lescure, 1969). In A. suum, glucose is catabolized in the cytosol to PEP accompanied by the reduction of NADP P to NADH. COR2R fixation into PEP allows for the formation of OAA that is enhanced by the low activity level of pyruvate kinase in the muscle mitochondria of A. suum. Malate dehydrogenase reduces OAA to malate, with concomitant regeneration of cytosolic NADP P. Cytosolic malate serves as the substrate for the mitochondrial dismutation reaction. The mitochondrial, NADP P-linked malic enzyme oxidizes half of the malate to pyruvate and COR2 Rwith concomitant NADH formation. The rest of the mitochondrial malate is converted to fumarate by fumarase, with fumarate serving as the terminal electron acceptor. The reduction of fumarate to succinate via the NADH-coupled fumarate reductase completes the anaerobic dismutation reaction of ascarid muscle mitochondria, thereby allowing ATP generation (Saz and Lescure, 1969; Fioravanti and Saz, 1980; Saz, 1981). As noted above, the NADPH NADP P transhydrogenase is essential to the anaerobic metabolism of H. diminuta inasmuch as this system is essential for electron transport in H. diminuta; without the transhydrogenase enzyme, fumarate would not ultimately be reduced to succinate. The mitochondrial NADPH NADP P transhydrogenase system is inner membrane-

17 9 associated and the active sites of the transhydrogenase face the matrix side of the mitochondrial inner membrane (McKelvey and Fioravanti, 1985).

18 10 Figure 4: Pathway of carbohydrate dissimilation in Ascaris muscle (Rew and Saz, 1974).

19 11 A reversible hydride ion transfer is catalyzed by the H. diminuta pyridine nucleotide transhydrogenase (Fioravanti and Saz, 1976). The NADH-forming reaction can be considered to be the forward reaction in the helminth, while the NADPH-forming reaction is considered to be the reverse reaction. In the presence of ATP, the transhydrogenase catalyzes an energy- linked reduction of NADPP P by NADH (Park and Fioravanti, 2006). This energy linkage also occurs via the electron transport-coupled oxidation of NADH. Lastly, the H. diminuta transhydrogenase can also serve as a transmembrane proton translocator and, thus, may act as an alternate site for anaerobic phosphorylation in its catalysis of the NADPH NAD reaction (Mercer-Haines and Fioravanti, 2008). The ascarid system apparently lacks an NADPH NADP P transhydrogenase which is consistent with the nematode having an NADP P-preferring mitochondrial malic enzyme (Saz and Lescure, 1969; Fioravanti and Saz, 1976; Fioravanti, 1981). Thus, reducing power required for the electron transport-dependent reduction of fumarate to succinate in A. suum is produced in the form of NADH (Saz and Lescure, 1969). The intramitochondrial localization of the malic enzyme in A. suum has been the subject of some controversy. Rew and Saz (1974) reported that the predominant localization of the ascarid mitochondrial malic enzyme as well as fumarase is the intermembrane space. As reduction of fumarate to succinate is thought to occur on the matrix side of the mitochondrial inner membrane (IM) (Lehninger, 1951), a mechanism for the transmembrane transfer of reducing power from the intermembrane space (IMS) to the matrix compartment would be required and fumarate would need to traverse the IM. Fioravanti and Saz (1976) noted the

20 presence of an NADH NADP P transhydrogenation system associated with the IM of both adult H. diminuta and A. suum. Moreover, Köhler and Saz (1976) found that ascarid mitochondria are capable of oxidizing exogenous NADH without affecting intramitochondrial pyridine nucleotide content presumably via the action of the NADH NADP Psystem. However, Köhler (1977) and Köhler et al. (1983) subsequently reported that the malic enzyme is equally distributed between the IMS and matrix of A. suum mitochondria and that fumarate does not penetrate the IM of these organelles. Despite these conflicting reports, the NADH NADP P activity in both the H. diminuta and A. suum systems remains intriguing. 12 Flavonoids Flavonoids are polyphenolic compounds that are widely distributed in nature, and are especially found in fruits and vegetables (Buhler and Miranda, 2000). Flavonoids are divided into different categories based on their chemical structure: flavonols, flavones, flavanones, isoflavones, catechins, anthocyanidins, and chalcones (Buhler and Miranda, 2000). Flavonoids have many roles in plant, insect, and mammalian systems. In plants, they are the most important pigmentation factor, producing yellow, orange, and red coloration (Middleton et al., 2000). Flavonoids have also been found to have a role in UV filtration, symbiotic nitrogen filtration, and can act as a chemical messenger, physiological regulator, or a cell cycle inhibitor (Hollman et al., 1996). One of the best known properties of flavonoids in mammals is their antioxidant activity. However, flavonoids are also known for their anti-inflammatory, anti-allergic, hepatoprotective, antithrombic, antiviral, and anticarcinogenic activities (Middleton et al., 2000).

21 Quercetin is the most abundant and most consumed flavonoid (Buhler and Miranda, 2000) that is derived primarily from tea, apples, and onions (Middleton et al., 2000). 13 Flavonoids have been found to affect both mammalian and invertebrate enzyme systems. Some mammalian enzyme systems found to be affected (inhibited) by flavonoids (e.g., quercetin, chrysin, apigenin) are phospholipases AR2 R(PLAR2R)R Rand C (PLC), kinases, ATPases, aromatase, malate dehydrogenase, lactic dehydrogenase and pyruvate kinase (Middleton et al., 2000). In terms of flavonoid interactions with invertebrate systems, studies indicate that these substances affect insect development, reproduction, and mitochondrial P450 systems (Hodgson, 1985; Ahmad, et al., 1986; Reese and Holyoke, 1987; Simmonds, 2003). More specifically, studies with the tobacco hornworm, Manduca sexta, demonstrated effects of flavonoids on two crucial enzyme systems. Mitchell et al. (1993) found that a number of plant flavonoids exerted inhibitory/stimulatory effects on the mitochondrial ecdysone 20-monooxygenase (E20M) of the tobacco hornworm, M. sexta. This enzyme is required to produce the active molting hormone, viz., 20-hydroxyecdysone. The insect E20M displays NADPH specificity (Smith et al., 1979). Vandock et al. (2008) characterized a reversible NADPH NAD transhydrogenase in M. sexta mitochondria that would serve in the formation of the needed NADPH. Indeed, subsequent findings indicated that a number of the flavonoids that exerted dose-dependent effects on the M. sexta E20M system also affected this insect s NADPH NADP Ptranshydrogenase (Vandock, 2010).

22 14 Purpose of Research Relatively little data exist as to the effects of flavonoids on invertebrate enzyme systems. In M. sexta, the flavonoids examined affect mitochondrial reduced pyridine nucleotide- utilizing enzymes viz., NADPH-specific E20M and the NADPH NADP Ptranshydrogenase. With respect to parasitic helminths, information is lacking concerning the potential effects of flavonoids on pyridine nucleotide-utilizing mitochondrial systems. Given these considerations, experiments were undertaken to evaluate the effects of some plant flavonoids on mitochondrial, membrane-associated reduced pyridine nucleotide utilizing systems of adult H. diminuta and A. suum. To this end the following membrane-associated systems were examined: NADPH NADP Ptranshydrogenase (not found in A. suum), NADH dehydrogenase, NADH cytochrome c reductase, and an NADH NADP Ptranshydrogenation.

23 15 CHAPTER II: THE STUDY Introduction The adult intestinal cestode, Hymenolepis diminuta, is predominately anaerobic with respect to its energetics and accumulates succinate as the major end product of glucose utilization (Scheibel and Saz, 1966; Scheibel et al., 1968). While succinate is the major end product, lesser amounts of lactate and acetate also accumulate from carbohydrate dissimilation (Scheibel and Saz, 1966; Scheibel et al., 1968). H. diminuta appears to be metabolically similar to the adult intestinal nematode Ascaris lumbricoides (Saz and Lescure, 1969; Saz et al., 1972). Phosphoenolpyruvate (PEP), arising from glycolysis, acts as a substrate for a COR2R-fixing reaction catalyzed by PEP carboxykinase, thereby resulting in oxaloacetate (OAA) formation. OAA is reduced to malate by an active, NADH-coupled malate dehydrogenase, thus supplying the mitochondrial substrate for H. diminuta. Malate, formed in the cytosol, serves as the anaerobic mitochondrial substrate, which is used for the physiologically anaerobic, electron transport-coupled, net generation of ATP. Within the mitochondrion, malate serves as the substrate for a dismutation reaction. The oxidative arm of the dismutation is catalyzed by an NADP-specific malic enzyme resulting in the formation of pyruvate, COR2R, and reducing power in the form of NADPH (Scheibel and Saz, 1966; Scheibel et al., 1968; Saz et al., 1972). However, the anaerobic, electron transport-coupled, and succinate-forming fumarate reductase utilizes NADH as the preferred reductant (Saz et al., 1972; Fioravanti, 1981). This dilemma is alleviated by the H. diminuta mitochondrial, inner membrane-associated NADPH NADP P transhydrogenase in its catalysis of hydride ion transfer from malic enzyme-produced NADPH to NADP P, thereby producing the needed NADH for anaerobic phosphorylation (Saz et al., 1972;

24 P transhydrogenase 16 Fioravanti and Saz, 1976; Fioravanti, 1981). This transhydrogenase is reversible, phospholipiddependent, energy- and non-energy-linked in terms of NADPH formation, and acts as a vital link in the anaerobic energetics of the cestode (Fioravanti and Saz, 1976; Fioravanti, 1981). As indicated, the accumulation of succinate in H. diminuta occurs via sequences similar to those of A. lumbricoides (Fioravanti and Saz, 1980). However, the need for an NADPH NADP in A. lumbricoides is not apparent because in A. lumbricoides malate is oxidized to pyruvate, with COR2R liberation and NADH formation,r Rvia an NAD-linked malic enzyme (Saz and Lescure, 1969). Malate then is converted to fumarate, which is then reduced to succinate in a manner similar to that of H. diminuta (Fioravanti and Saz, 1980). Flavonoids are polyphenolic compounds that are widespread in nature and are well noted in fruits and vegetables (Buhler and Miranda, 2000). These compounds have been demonstrated to affect both mammalian and invertebrate enzyme systems. Some mammalian enzyme systems inhibited by flavonoids (e.g., quercetin, chrysin, apigenin) are phospholipases AR2 Rand C, kinases, ATPases, aromatase, malate dehydrogenase, lactic dehydrogenase and pyruvate kinase (Middleton et al., 2000). In terms of interactions with invertebrate systems, flavonoids affect insect development, reproduction, and mitochondrial P450 systems (Hodgson, 1985; Ahmad et al., 1986; Reese and Holyoke, 1987; Mitchell et al., 1993; Simmonds, 2003). Indeed, studies with the tobacco hornworm, Manduca sexta, demonstrated flavonoid effects on two crucial enzyme systems. Mitchell et al. (1993) found that a number of plant flavonoids exerted inhibitory/stimulatory effects on the mitochondrial, NADPH-specific ecdysone 20-

25 P transhydrogenase monooxygenase (E20M) of the tobacco hornworm, M. sexta, the enzyme needed to produce the active molting hormone, 20-hydroxyecdysone. Vandock et al. (2008) characterized a mitochondrial, reversible NADPH NADP P transhydrogenase in M. sexta that would serve in NADPH formation needed for E20M activity. Moreover, subsequent findings indicated that a number of the flavonoids that exerted dose-dependent effects on the M. sexta E20M system also affected this insect s NADPH NADP P transhydrogenase (Vandock, 2010; Vandock et al., 2012). 17 Clearly, relatively little data exist as to flavonoid effects on invertebrate enzyme systems. In M. sexta, the flavonoids examined affect mitochondrial, reduced pyridine nucleotide-utilizing enzymes viz., NADPH-dependent E20M and the NADPH NADP P transhydrogenase. More specifically, information is lacking concerning the potential effects of flavonoids on pyridine nucleotide-utilizing mitochondrial systems of the parasitic helminths. Given these considerations, evaluations of the effects of some plant flavonoids on mitochondrial, membraneassociated reduced pyridine nucleotide utilizing systems of adult H. diminuta and A. suum were undertaken. Thus, the following membrane-associated systems were examined: NADPH NADP (not found in A. suum), NADH dehydrogenase, NADH cytochrome c reductase, and an NADH NADP P transhydrogenation.

26 18 Materials and Methods Mitochondrial preparations and Hymenolepis diminuta propagation The Hymenolepis diminuta life cycle was maintained employing the flour beetle, Tribolium confusum, as the intermediate host. Adult H. diminuta were obtained from experimentally infected male or female Sprague-Dawley rats (10 cysticercoids/animal) 21 or more days post-infection (Fioravanti and Saz, 1976). The worms were minced in mitochondrial medium consisting of 240 mm sucrose, 10 mm Tris (hydroxymethylaminomethane)-hcl (ph 7.5), 15 mm ethylenediaminetetraacetic acid (EDTA), and 0.15% bovine serum albumin (BSA) at a ratio of approximately 10 ml medium/g tissue. Thereafter, H. diminuta tissue was homogenized using a motorized Potter-Elvehjem homogenizer with a Teflon pestle. Homogenates were subjected to centrifugation at 482 x g for 10 min to remove cellular debris. Resulting supernatant fractions were centrifuged at 9200 x g for 30 min to isolate mitochondria. Mitochondria were washed in mitochondrial medium and centrifuged for an additional 30 min at 9200 x g. The washed organelles, suspended in mitochondrial medium, were subjected to 4 5 sec sonication bursts with 30 sec cooling intervals employing a Branson sonicator, equipped with a microtip at a power setting of 20 W. Mitochondrial membranes were isolated by centrifugation at 257,320 x g for 60 min and thereafter suspended in mitochondrial medium. Adult Ascaris suum were obtained from J. H. Routh Packing in Sandusky, Ohio. Muscle tissue was dissected predominantly from female ascarids and mitochondria and mitochondrial membranes were prepared after Fioravanti and Saz (1976) as described here for adult H. diminuta.

27 P transhydrogenase 19 All extractions were performed at 4P 0 PC. Both cestode and nematode membranes were 0 suspended in mitochondrial medium and stored at -20P PC. Isolated mitochondrial membranes served as the source of enzyme activities. Enzyme assays NADPH NADP activity was assessed spectrophotometrically by following the reduction of acetylpyridine NADP P (AcPyADP P) at a wavelength of 375 nm (Fioravanti and Saz, 1976; Fioravanti, 1981). In addition to enzyme and 1.9% ethanol, the 1 ml assay volume contained the following in µmoles: potassium phosphate (ph 7.5) 100; rotenone, 25; NADPH, 0.25; and AcPyADP P, 0.6. The NADH NADP P transhydrogenation was assayed similarly by measuring AcPyADP P reduction at 375 nm with the exceptions that the 1 ml volume contained 100 µmoles potassium phosphate (ph 7.5 for H. diminuta; ph 6.5 for A. suum) and 0.25 µmoles NADH (Fioravanti and Saz, 1976; Fioravanti, 1981). NADH dehydrogenase was assessed spectrophotometrically by measuring ferricyanide reduction at 410 nm after Walker and Fioravanti (1995). In addition to enzyme and 1.9% ethanol, the 1 ml assay volume contained the following in µmoles: potassium phosphate (ph 7.5 for H. diminuta; ph 6.5 for A. suum) 100; rotenone, 25; ferricyanide, 0.03; and NADH NADH cytochrome c reductase was evaluated by measuring the reduction of cytochrome c at a wavelength of 550 nm (Kim and Fioravanti, 1985). Aside from 1.9% ethanol and 0.3 mg of cytochrome c, the 1 ml assay volume contained the following in µmoles: potassium phosphate (ph 7.5 in H. diminuta; ph 6.5 in A. suum), 100; and NADH, 0.25.

28 In all instances, flavonoids were dispensed in ethanol and rotenone such that ethanol was present at 1.9% and the flavonoid was present at the indicated concentration in the 1 ml assay 0 volume. Enzyme assessments were performed at 25P PC employing a Beckman Model 25 dualbeam spectrophotometer. Protein content was determined by the Bradford method (1976) using BSA as the standard. 20 Statistical analyses Statistical analyses were accomplished using JMP 10 (SAS) software through one-way ANOVA and Tukey s post-hoc comparison test. Significance was determined when p < Materials NADH, AcPyADP P, cytochrome c, rotenone, EDTA, and flavonoids were obtained from Sigma-Aldrich Chemical Company, St. Louis, MO. NADPH was purchased from Calbiochem, San Diego, CA. The flavonoids were a generous gift from Dr. Kurt Vandock, Department of Biology, Houghton College, N.Y. Sucrose, sodium phosphate, monobasic and ferricyanide were purchased from Fisher Scientific, Pittsburg, PA. Potassium phosphate, monobasic and dibasic, was obtained from EMB Chemicals, Inc., Gibbstown, NJ. Tris was purchased from Amresco, Solon, OH. Crystalline BSA was obtained from ICN Biomedical, Inc, Irvine, CA. Ethanol was purchased from Pharmco-Aaper, Brookfield, CT. Bio-Rad protein assay reagent was obtained from Bio-Rad Laboratories, Richmond, CA.

29 21 Results As part of the assessments of flavonoids on helminth enzymatic activities, appropriate controls were performed to evaluate the effects of flavonoids on substrates and acceptors in the absence of mitochondrial membranes. These evaluations indicated that the flavonoids were essentially without effect on substrates and acceptors. Moreover, with the exception of the NADH cytochrome c reductase, rotenone was present in all assays to prevent electron transportdependent reduced pyridine nucleotide oxidation via mitochondrial NADH oxidase (Walker and Fioravanti, 1995). In the case of NADH cytochrome c reductase, the presence of rotenone would inhibit cytochrome reduction (Fioravanti and Kim, 1983) and, thus, ethanol was present while rotenone was omitted. Hymenolepis diminuta The effects of flavonoids on the Hymenolepis diminuta NADPH NADP P transhydrogenase were evaluated and the results are presented in Figure 5. As noted in this figure, the flavonoids evaluated did not statistically alter transhydrogenase activity when compared to corresponding controls. However, a tendency towards activity stimulation occurred with the juglone additive (~ 400%). Furthermore, the addition of plumbagin to the assay system significantly affected enzyme activity at concentrations of 10 P -7-3 Pto 10P P M when compared with one another. Indeed, the tendency observed suggested an increase in transhydrogenase activity at the highest concentration of plumbagin evaluated (~400%), despite apparent decreased activity at lower concentrations (Fig. 5).

30 Figure 5. Significant difference compared to other concentrations of the same flavonoid. Control activity was 35.5 ± 2.9 nmol/min/mg; N=47. Number of samples measured at each concentration for each flavonoid ranged from one to 12. Activities expressed reduction of acetylpyridine NAD. Rotenone was dispensed in ethanolic solution such that the rotenone content was 25 µm and the ethanol was 1.9% mg protein was used for assays. 22

31 23 While all of the flavonoids were not assessed with respect to the H. diminuta NADH dehydrogenase activity, the effects of plumbagin were examined and these data are presented in Figure 6. At the lower flavonoid concentrations, i.e., 10 P -8 P, 10 P -7 PM, significant reductions in activity were noted when compared to corresponding controls. Although there were no significant differences between the various concentrations, a tendency towards increased -4 dehydrogenase activity was observed, approaching ~100% of controls, at 10P PM plumbagin (Fig. 6). Potential flavonoid effects also were examined with respect to the cestode NADH cytochrome c reductase and these results are given in Figure 7. Whereas not all flavonoids at each concentration were evaluated nor were all flavonoids apparently effective in altering activity, two of the additives examined, viz., chrysin and juglone, did display apparent effectiveness in stimulating NADH cytochrome c reductase. Significant differences were -6-4 observed amongst juglone concentrations of 10P Pto 10P PM with a marked increase in enzyme -4 activity (~1400 %) at 10P PM.P PFurthermore, a tendency towards stimulation in activity (~700 %) -4 was noted with chrysin at 10P P M (Fig. 7). The data obtained when H. diminuta NADH NADP P transhydrogenation activity was assessed in the presence of increasing concentrations of flavonoids is presented in Figure 8. Although not all flavonoids at all concentrations were evaluated, and statistically significant differences between flavonoid-treated versus controls were not found, the NADH NADP P transhydrogenation displayed a potential tendency of significance was chrysin and juglone.

32 From 10P P to 10P PM chrysin, potential stimulations were noted (~ 120 %) as were suggested -7-3 stimulations with juglone from 10P P to 10P P M (~ 140 %) (Fig. 8).

33 Figure 6. * Significantly different from control. Control activity was ± 48.8 nmol/min/mg; N=6. Number of samples measured at each concentration ranged from one to three. Activities expressed reduction of ferricyanide. Rotenone was dispensed in ethanolic solution such that the rotenone content was 25 µm and the ethanol was 1.9%. 0.7 mg protein was used for assays. 25

34 Figure 7. Significant difference from other concentrations of same flavonoid. * Significant difference from control. Control activity was 72.3± 4.9 nmol/min/mg; N=13. Number of samples measured at each concentration for each flavonoid ranged from two to three. Activities expressed reduction of cytochrome c. Ethanol was dispensed such that the ethanol was 1.9%. 0.1 mg protein was used for assays. 26

35 Figure 8. Control activity was 74.0 ± 4.2 nmol/min/mg; N=26. Number of samples measured at each concentration for each flavonoid ranged from two to three. Activities expressed reduction of acetylpyridine NAD. Rotenone was dispensed in ethanolic solution such that the rotenone content was 25 µm and the ethanol was 1.9% mg protein was used for assays. 27

36 28 Ascaris suum The effects of flavonoid additions on the corresponding Ascaris suum mitochondrial, reduced pyridine-nucleotide utilizing activities were evaluated and the data are presented here. Whereas all of the flavonoids at all concentrations were not evaluated nor were all flavonoids effective with respect to the A. suum NADH dehydrogenase activity, two of the flavonoids examined, viz., morin and plumbagin exerted significant effects (Fig. 9). At the lower concentrations of morin, in the range of 10P P to 10P PM significant stimulations of activity as compared to controls was noted as was differences in effects of concentrations with one another. In addition, there was a significant reduction of activity from the maximum stimulation noted that appeared dose dependent. On the other hand, plumbagin displayed a tendency towards -5 stimulation of the ascarid NADH dehydrogenase with a significant peak in activity at 10P PM as compared to corresponding controls (Fig. 9). The effects of the flavonoids on the nematode mitochondrial NADH cytochrome c reductase were examined and the results obtained are given in Figure 10. As indicated, not all -7-3 flavonoids were studied and the concentration range was 10P P to 10P P M. Seemingly, the only flavonoid affecting the reductase was juglone. Significant differences between juglone concentrations were noted in the concentration range of 10P P to 10P P M. Indeed, at 10P P M, juglone exerted a marked and significant stimulation of activity in an apparent dose-dependent fashion (Fig.10).

37 Figure 9. Significantly different from other concentrations of same flavonoid. * Significantly different from control. Control activity was ± nmol/min/mg; N=6. Number of samples measured at each concentration for each flavonoid ranged from two to four. Activities express the reduction of ferricyanide. Rotenone was dispensed in ethanolic solution such that the rotenone content was 25 µm and the ethanol was 1.9% mg protein was used for assays. 29

38 Figure 10. Significantly different from other concentrations of same flavonoid. * Significantly difference compared to control. Control activity was 8.6 ± 1.9 nmol/min/mg; N=18. Number of samples measured at each concentration for each flavonoid ranged from one to five. Activities express the reduction of cytochrome c. Ethanol was dispensed such that the ethanol was 1.9% mg protein was used for assays. 30

39 P transhydrogenation 31 As with H. diminuta, an evaluation of the effects of the flavonoids on the A. suum NADH NADP reaction was pursued and the results obtained are given in Figure 11. Not all of the flavonoids were evaluated at each concentration and the concentration -7-3 range of flavonoids was 10P P to 10P P M. Although not every flavonoid affected activity, three, viz., chrysin, juglone and plumbagin, appeared to exert effects on the transhydrogenation. Both -3 chrysin and juglone at 10P P M significantly inhibited activity (~70 and 60 %, respectively). Indeed, juglone displayed some significant differences in comparisons of concentrations. -3 Moreover, a tendency towards stimulation (~140 %) by plumbagin was apparent at 10P PM (Fig. 11).

40 Figure 11. Significantly different from other concentrations of same flavonoid. * Significantly different from control. Control activity was ± 18.9 nmol/min/mg; N=27. Number of samples measured at each concentration for each flavonoid ranged from two to four. Activities express the reduction of acetylpyridine NAD. Rotenone was dispensed in ethanolic solution such that the rotenone content was 25 µm and the ethanol was 1.9% mg protein was used for assays. 32

41 33 Discussion A number of flavonoids have been found to affect both vertebrate and invertebrate systems (Middleton et al., 2000). With respect to invertebrates, and more specifically insect systems, these compounds have been found to affect reproduction and development (Reese and Holyoke, 1987; Simmonds, 2003). Indeed, it has been demonstrated that plant flavonoids appear to directly affect reduced pyridine nucleotide-utilizing systems, viz., the mitochondrial NADPH- specific ecdysone 20-monooxygenase (E20M) and the NADPH NADP Ptranshydrogenase of the tobacco hornworm, Manduca sexta (Mitchell et al., 1993; Vandock et al., 2012). The former enzyme acts to convert ecdysone to the physiologically active hormone whereas the latter enzyme seems to provide some NADPH for active hormone formation (Smith et al., 1979; Smith, 1980; Vandock et al., 2008; 2010). Because no studies existed as to the potential direct effects of flavonoids on parasitic helminth mitochondrial NADPH NADP P transhydrogenase and other reduced pyridine nucleotide-coupled enzyme systems (viz., mitochondrial, electron transport-linked NADH dehydrogenase, NADH-cytochrome c reductase and the NADH NADP P transhydrogenation reaction (Walker and Fioravanti, 1995; Fioravanti and Vandock, 2009; Holoweicki, 2009)) evaluations of flavonoid effects on the Hymenolepis diminuta and Ascaris suum membrane-associated systems were examined. Accordingly, the effects of some of the flavonoids examined in terms of the insect model M. sexta, i.e., chrysin, quercetin, morin, and juglone (Vandock et al., 2012) were studied. Although some time limitations, based on the acquisition of parasite material as well as flavonoids and the retirement of Dr. Fioravanti,

42 34 resulted in not all the flavonoids at all concentrations being assessed, sufficient data were obtained suggesting that some flavonoids can affect the helminth mitochondrial systems. Vandock et al. (2012) reported that the M. sexta transhydrogenase systems of midgut and fat body mitochondria responded to the direct presence of plant flavonoids. Whereas midgut mitochondrial NADPH NADP Pactivity was inhibited by chrysin, quercetin, morin, juglone, and myricetin, the fat body enzyme was inhibited by chrysin and juglone, but stimulated by quercetin, morin and myricetin. Certainly, the suggestion that the corresponding H. diminuta enzyme differs from the insect system was noted inasmuch as the flavonoids examined did not cause significant differences when compared to controls. However, it is of note that a tendency towards inhibition was seen with chrysin and the helminth enzyme tended to be stimulated in the presence of juglone and plumbagin. In their study of M. sexta mitochondria, Vandock et al. (2012) also examined flavonoid effects on two electron transport coupled activities, viz., NADH oxidase and succinate dehydrogenase. In the Manduca study, the midgut systems were inhibited by chrysin, morin, juglone, and myricetin, although quercetin was without effect. Likewise, fat body NADH oxidase and succinate dehydrogenase were inhibited by chrysin and juglone, but stimulated by morin and myricetin. In the present study only the effect of plumbagin on the H. diminuta NADH dehydrogenase was examined while a more complete study of the corresponding ascarid system was undertaken. With respect to H. diminuta, plumbagin significantly inhibited activity at lower concentrations, but with an increase in concentration a tendency towards stimulation was suggested. On the other hand, the ascarid NADH dehydrogenase was significantly

43 35 stimulated by morin at lower concentrations and a significant lessening of activity was apparent with increasing morin concentrations in a dose-dependent fashion. Furthermore, plumbagin seemingly stimulated activity with a significant stimulation noted at next to the highest concentration examined. Thus, the helminth systems were similar to one another in terms of plumbagin effects and the ascarid system was similar to M. sexta in terms of morin stimulation. A comparison of H. diminuta and A. suum NADH cytochrome c reductase responses to plant flavonoids indicated that in both parasitic helminths juglone acts as a significant stimulator of activity. Accordingly, a similarity in the two anaerobic helminths was apparent. However, these stimulatory responses differed from what was noted with respect to M. sexta electrontransport linked systems examined wherein juglone acted as an inhibitor (Vandock et al., 2012). Both the H. diminuta and A. suum systems display a membrane-associated NADH NADP Ptranshydrogenation activity. A comparison of the cestode and nematode activities revealed the following: Whereas the H. diminuta reaction was not significantly affected by the presence of flavonoids, a tendency towards stimulation by plumbagin was noted as well as the potential for morin inhibition and some stimulation by chrysin. Moreover, juglone tended towards stimulation. In contrast, the nematode transhydrogenation was significantly inhibited by chrysin and juglone. As with the cestode reaction, the nematode transhydrogenation was leaning towards stimulation in the presence of plumbagin. As indicated, the M. sexta E20M activities are affected by plant flavonoids (Mitchell et al., 1993). The midgut E20M is inhibited by chrysin, and following stimulation at lower concentrations, morin and quercetin were inhibitory. However, with respect to fat body E20M,

44 36 chrysin was without effect while morin and quercetin were quite stimulatory at lower concentrations prior to displaying inhibition. Thus, as with the Manduca transhydrogenase, both similarities and differences were noted in comparisons of the E20M systems and the helminth activities. Clearly, further studies with parasitic helminth systems relating to effects of flavonoids on the activities examined are warranted. Nonetheless, the data presented with respect to the NADPH NADP Pof H. diminuta supports the notion that this mitochondrial system differs from the corresponding systems of a free living organism. The other membrane associated helminth systems reflect components of the mitochondrial, anaerobic electron transport systems, i.e., NADH dehydrogenase, Complex I (Walker and Fioravanti, 1995); NADH cytochrome c reductase, Complex I-III (as reviewed by Fioravanti and Vandock, 2009); NADH NADP P Complex I and the pyruvate dehydrogenase complex (Walker and Fioravanti, 1995; Holoweicki, 2009). The data obtained suggest the following: A similarity of the helminth NADH dehydrogenases in terms of plumbagin stimulation; a similarity of the NADH cytochrome c reductases with respect to juglone stimulation; and differences in juglone effects, i.e., possibly stimulatory in the cestode and inhibitory in the nematode. By way of comparison, the NADH oxidase activities of M. sexta midgut and fat body, that would reflect mitochondrial Complex I-IV, and succinate dehydrogenase, that would reflect Complex II, were mostly inhibited by the flavonoids evaluated (chrysin, morin, juglone, myricetin). Certainly, a difference in the anaerobic vs. aerobic electron transport systems was apparent. Given the possible impact of the flavonoids on helminth

45 mitochondrial systems, the possibility that they or other flavonoids can be of chemotherapeutic benefit in terms of the parasitic helminths is worthy of consideration. 37 For the first time, plant flavonoids have been found to affect crucial mitochondrial systems needed for anaerobic energy generation in two model parasitic helminths. Aside from a more comprehensive examination of flavonoid effects on H. diminuta and A. suum, the present study sets the framework for future studies. Thus, examinations of flavonoid effects on other helminth parasite anaerobic mitochondria are warranted as are comparative studies with mammalian mitochondrial systems. In addition, a more comprehensive survey of flavonoids and studies as to mechanisms for flavonoid stimulation/inhibition will be of interest..

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47 39 Fioravanti, C. F. and H. J. Saz Energy metabolism of adult Hymenolepis diminuta. In: Biology of the Tapeworm Hymenolepis diminuta (Arai, H. P., ed.), pp , Academic Press, New York. Fioravanti, C. F. and K. P. Vandock Transhydrogenase and the anaerobic mitochondrial metabolism of adult Hymenolepis diminuta. Parasitology. 137: 3, Hodgson, E., G. A. Kerkut, L. I. Gilbert TComprehensive insect physiology, biochemistry and pharmacology0t. Oxford: Pergamon Press. Hollman, P. C. H., M. V. D. Gaag, M. J. B. Mengelers, J. M. P. Van Trip, J. H. M. De Vries, and M. B. Katan Absorption and disposition kinetics of the dietary antioxidant quercetin in man. Free Radical Biology and Medicine. 21:5, Holowiecki, A Catalysis of mitochondrial NADH NAD transhydrogenation in adult Ascaris suum (nematoda). Bowling Green State University. OhioLINK ETD Center. Retrieved from 1TUhttp://etd.ohiolink.edu/send-pdf.cgi/Holowiecki%20Andrew.pdf?bgsu U1T Horie, Y. and W. Chefurka The distribution and properties of transhydrogenases in insect tissues. Comparative Biochemistry and Physiology. 8: Johnstone, C. (2000, February 21). Parasites and parasitic diseases of domestic animals. Retrieved from

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