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Parasitology Internationa

Cyanide-resistant respiration in Taenia crassiceps metacestode

(cysticerci) is explained by the H2O2-producing side-reaction of

respiratory complex I with O2

I. Patricia del Arenala,*, M. Esther Rubiob, Jorge Ramırezc, Juan L. Rendona,

J. Edgardo Escamillac

aDepartamento de Bioquımica, Facultad de Medicina, Universidad Nacional Autonoma de Mexico, Apartado postal 70-159, Mexico 04510 D.F., MexicobDepartamento de Fisiologıa, Instituto Nacional de Cardiologıa ‘‘Ignacio Chavez’’, Mexico 14080 D.F., Mexico

cInstituto de Fisiologıa Celular, Universidad Nacional Autonoma de Mexico, Apartado postal 70-242, Mexico 04510 D.F., Mexico

Received 9 August 2004; accepted 14 April 2005

Available online 14 June 2005

Abstract

The nature of the cyanide-resistant respiration of Taenia crassiceps metacestode was studied. Mitochondrial respiration with NADH as

substrate was partially inhibited by rotenone, cyanide and antimycin in decreasing order of effectiveness. In contrast, respiration with

succinate or ascorbate plus N,N,NV,NV-tetramethyl-p-phenylenediamine (TMPD) was more sensitive to antimycin and cyanide. The saturation

kinetics for O2 with NADH as substrate showed two components, which exhibited different oxygen affinities. The high-O2-affinity system

(Km app=1.5 AM) was abolished by low cyanide concentration; it corresponded to cytochrome aa3. The low-O2-affinity system (Km app=120

AM) was resistant to cyanide. Similar O2 saturation kinetics, using succinate or ascorbate–TMPD as electron donor, showed only the high-

O2-affinity cyanide-sensitive component. Horse cytochrome c increased 2–3 times the rate of electron flow across the cyanide-sensitive

pathway and the contribution of the cyanide-resistant route became negligible. Mitochondrial NADH respiration produced significant

amounts of H2O2 (at least 10% of the total O2 uptake). Bovine catalase and horse heart cytochrome c prevented the production and/or

accumulation of H2O2. Production of H2O2 by endogenous respiration was detected in whole cysticerci using rhodamine as fluorescent

sensor. Thus, the CN-resistant and low-O2-affinity respiration results mainly from a spurious reaction of the respiratory complex I with O2,

producing H2O2. The meaning of this reaction in the microaerobic habitat of the parasite is discussed.

D 2005 Elsevier Ireland Ltd. All rights reserved.

Keywords: Helminth; Respiratory chain; Cytochrome o; Respiratory inhibitors; Hydrogen peroxide; Antioxidant enzymes

1. Introduction

The respiratory systems of helminths are more diverse

and complex than those of their hosts. Differences in the

respiratory system exist among many helminth species

and, in many cases, in different stages of their life cycles

[1,2]. Parasites, such as Ascaris [3,4], Moniezia [5] and

filaria [6,7] have been extensively studied. Parasites

exhibit aerobic and anaerobic metabolisms with succinate

1383-5769/$ - see front matter D 2005 Elsevier Ireland Ltd. All rights reserved.

doi:10.1016/j.parint.2005.04.003

* Corresponding author. Tel.: +52 55 56232169; fax: +52 55 56162419.

E-mail address: darenal@bq.unam.mx (I.P. del Arenal).

as the product of the fumarate reductase reaction [8,9].

Nutrient sources, CO2 and oxygen tension play an

important role in habitat variations during the parasite’s

alternate lifestyles.

Two properties of the energy metabolism of helminths

have been highlighted: the high resistance of respiration to

the classical respiratory inhibitors, particularly cyanide

[3,10], and the production of significant amounts of hydro-

gen peroxide by their mitochondria [11,12]. A putative type

o cytochrome has been frequently implicated in the

inhibitor-resistant respiration. Hence, alternative respiratory

chains with different terminal oxidases have been proposed

[13,14]. Production of H2O2 has also been reported as a by-

l 54 (2005) 185 – 193

I.P. del Arenal et al. / Parasitology International 54 (2005) 185–193186

product of cytochrome o oxidase [10,11,15] and, in

Hymenolepis diminuta, H2O2 production has also been

associated to fumarate reductase activity [16].

We previously detected cyanide-resistant respiration in

the mitochondrial fraction of Taenia crassiceps metaces-

tode, suggesting the existence of an alternative respiratory

pathway containing a cyanide-resistant terminal oxidase

[17]. Carbon monoxide difference spectra of mitochondria

reduced by substrate or dithionite showed the expected

cytochrome a3–CO adduct. In addition, these spectra

suggested the presence of a cytochrome o –CO-type

complex. However, haem O in metacestode mitochondria

was not detected by HPLC analysis in the mitochondrial

membrane fraction. Moreover, the spectral analysis of the

CO/O2 ligand exchange after photodissociation at subzero

temperatures did not reveal the presence of an additional

oxidase besides cytochrome aa3 [17].

To elucidate the nature of the cyanide-resistant respira-

tion in cysticerci mitochondria, we analysed aerobic

respiration in the whole organism and isolated mitochon-

dria, using different substrates and site-specific inhibitors.

Our results indicate that cysticerci mitochondria build up

high levels of hydrogen peroxide during respiration and that

respiratory complex I is the major site for H2O2 production.

It was also found that cyanide abolishes the activity of

cytochrome c oxidase without affecting the spurious

reaction of reduced complex I with oxygen to produce

H2O2. Additionally, it was found that the enzymatic

activities involved in the elimination of H2O2 in cysticerci

tissues, such as catalase, cytochrome c peroxidase and

glutathione peroxidase, were markedly low, causing an

accumulation of H2O2.

2. Materials and methods

2.1. The parasite

Female BALB/c mice were inoculated intraperitoneally

with 15 cysticerci of T. crassiceps HYG strain. Six to eight

months later, cysticerci were recovered from the peritoneal

cavity [18] and thoroughly washed with 10 mM phosphate

buffered saline (PBS), pH 7.4.

2.2. Preparation of mitochondria

Mitochondrial total fractions (tegument plus cellular

parenchyma) from the cysticerci were obtained by a

combination of previously reported methods [19]. Briefly,

to obtain tegument syncytium mitochondria, cysticerci

were resuspended in an equal volume of ice-cold buffer

containing 10 mM Hepes, 250 mM sucrose, 2 mM EGTA,

0.1% fatty acid-free albumin, pH 7.4 (BM buffer), plus 86

AM phenylmethylsulfonyl fluoride (PMSF) and 0.2%

saponin (Saponaria species). After incubation for 4 min

in an ice bath, the suspension was centrifuged at 180 �g

for 10 min and the supernatant containing the tegument

syncytium mitochondria was saved. The 180 �g pellet

containing the carcass (parenchyma) was resuspended in

BM-buffer plus 86 AM PMSF and mechanically disrupted

with a motor-driven Teflon pestle of a Potter–Elvehjen

homogeniser as described by Zenka and Prokopic [20].

The homogenate was subjected to a differential centrifu-

gation sequence at 180, 1200 and 14,500 �g for 10 min

each. The tegument syncytium (180 �g supernatant) and

the carcass (14,500 �g pellet) were mixed and washed

twice with BM-buffer. The final pellet contained the total

mitochondrial fraction. Centrifugations were always per-

formed at 4 -C. The mitochondrial fractions were stored at

�45 -C until use.

Liver mitochondria from Wistar adult rats were

prepared following the method of Martınez et al. [21].

The homogenate was prepared in 250 mM sucrose plus

1.0 mM EDTA, adjusted to pH 7.3 with Tris–HCl.

Purified mitochondria were resuspended in the same

homogenisation medium and stored at �45 -C.Before use, stored mitochondria were disrupted by

sonication (20 s) in a Soniprep 150 sonifier at one-half of

the maximal output. Protein was determined by the method

of Markwell et al. [22] using bovine serum albumin as

standard.

2.3. Enzyme activity assays

The activities of NADH, succinate and ascorbate–

TMPD oxidases were measured with a Clark-type O2

electrode using an YSI-53 oxygen monitor and ultrafine,

high-sensitivity Teflon membranes (Yellow Springs Instru-

ments, Ohio, USA). Measurements were performed at 30

-C in 1.8 ml of 10 mM Hepes buffer, pH 7.4, plus 0.5

mM EDTA and 1.0 mg of mitochondrial protein.

Substrate and inhibitor concentrations are indicated in

the figures. The hydrophobic inhibitors were previously

dissolved in absolute ethanol as a carrier-solvent and

preincubated with the mitochondrial samples (15 min at

37 -C) before starting the reaction by the addition of the

substrate. The final ethanol concentrations had no effect

on the respiration kinetics.

The respiratory activity of whole cysticerci with endog-

enous substrates was measured at 37 -C by the polaro-

graphic method in 1.8 ml of PBS, containing 79 mg of

cysticerci (dry weight).

Endogenous catalase activity was determined in the

whole cysticerci homogenate and the mitochondrial frac-

tion in 1.8 ml of N2-saturated 10 mM potassium phosphate

buffer (pH 7.4) containing 1–5 mg of sample protein

according to the method of Goldstein [23]. The reaction

was started by adding 16 mM H2O2 (final concentration);

oxygen evolution was followed with a Clark-type elec-

trode. True catalase activity was considered as the fraction

of the activity that was inhibited by 8 mM 3-amino-1,2,4-

triazole [24].

I.P. del Arenal et al. / Parasitology International 54 (2005) 185–193 187

Glutathione peroxidase activity was determined accord-

ing to the coupled method of Paglia and Valentine [25] in

2.0 ml of 100 mM Tris–HCl buffer (pH 7.8) containing 3

mM EDTA, 6 mM GSH, 0.24 mM NADPH and 0.5 Ag of

spinach glutathione reductase. The reaction was started by

the addition of H2O2 up to a final concentration of 16 AMand NADPH oxidation was measured at 340 nm. The

NADPH molar absorptivity (6.22�103 M�1 cm�1) was

used to calculate the specific activity.

2.4. Microscopy

For transmission electron microscopy, cysticerci were

fixed in 3% (v /v) glutaraldehyde for 2 h and post-fixed in

2% (w /v) osmium tetraoxide. The fixed preparation was

sectioned and embedded in Epon. Sections were stained in

2% (w /v) uranyl acetate, pH 4.8, and lead citrate [26].

A stock solution of 57.8 mM dihydrorhodamine 123

(DHR from Molecular Probes) was dissolved in dime-

thylformamide and aliquots were stored in the dark at

�20 -C. Fresh cysticerci were incubated at a 1 :1000

dilution of the DHR stock solution or at a 1 :10 dilution

of the 1.9 AM MitoTraker CMXros (Molecular Probes)

stock solution. The stained cysticerci were imaged with a

laser scanning confocal microscope (Biorad MRC 1024

Hercules, CA, USA). Excitation and emission settings

were 488 and 522 nm for Rhodamine and 568 and 605

nm for MitoTraker.

2.5. Determination of hydrogen peroxide

Hydrogen peroxide produced during mitochondrial

respiration was polarographically measured with a Clark-

type electrode as the amount of O2 evolved by bovine liver

catalase (60 units) added at defined times after respiration

was started with exogenous substrate. Alternatively, H2O2

was spectroscopically determined by the formation of a

peroxide-dependent ferrithiocyanate complex [27] as fol-

lows. The reaction samples (1 ml) contained 100 mM

Tris–HCl, pH 7.5, 1.0 mg of sample protein and the

substrate concentrations as shown in Table 1. Reference

blanks contained all reaction constituents, except the

electron donor substrate. After 10 min of incubation at

30 -C, the reaction was stopped by adding 0.1 ml of 40%

(w /v) trichloroacetic acid to the samples and reference

Table 1

Respiratory activity and H2O2 production by T. crassiceps cysticerci mitochondri

NADH Succinate

Specific activitya H2O2 productionb Specific act

Control 93T18 (6) 4.5T0.8 (6) 51T6 (5)

10 AM rotenone 14T0.8 (6) 0.7T0.5 (4) ND

10 AM antimycin 37T4.2 (5) 4.0c 16T1.2 (5)

1 mM CN 44T8 (5) 3.7T1.3 (3) 4.3T0.7 (3)

Substrate concentration: 1 mM NADH, 15 mM succinate, 10 mM ascorbate, 15 AMnot done. MeanTS.E. Number of determinations is presented in parentheses. cAv

blanks. Then, the electron donor substrate was added to

blanks. Protein was removed by centrifugation at 14,500

�g for 15 min and 4 -C. Thereafter, 0.2 ml of 10 mM

ferrous ammonium sulfate was added to all supernatants.

Formation of the ferrithiocyanate complex was induced by

the addition of 0.1 ml of 2.5 M fresh potassium

thiocyanate. Spectrophotometric measurements were made

at 480 nm, 30 min after the addition of potassium

thiocyanate. To calculate the rates of H2O2 production,

the endogenous catalase activity of our preparations was

neglected.

3. Results and discussion

3.1. The respiratory pathways

O2 uptake by T. crassiceps mitochondria was studied in

the presence of substrates and site-specific inhibitors (Fig.

1). Inhibition of NADH oxidase by either rotenone,

antimycin A or KCN was biphasic; it showed a component

with high sensitivity to inhibitors and a component that was

clearly resistant to the inhibitors tested; 20%, 60%, and 30%

of the total NADH oxidase remained active in the presence

of high concentrations of rotenone, antimycin and cyanide,

respectively (Fig. 1A–C). This behaviour suggested the

presence of an alternative respiratory pathway, insensitive to

classical respiratory inhibitors.

Succinate-oxidase was inhibited by antimycin or cyanide

to a higher extent than NADH oxidase; however, the

inhibition profiles still showed two kinetic components

(Fig. 1B and C). Apparently, the inhibitors were acting on

the same component regardless of whether the substrate was

succinate or NADH. The ascorbate–TMPD mixture prefer-

entially feeds electrons to high potential c-type cyto-

chromes. Thus, as expected, cytochrome c oxidase in

cysticerci mitochondria was fully inhibited by 5 AM KCN

(Fig. 1C, inset) with a Ki =1.3 AM, which is in agreement

with the usual high sensitivity of type aa3 oxidases to

cyanide [28,29].

The O2 saturation kinetics showed that NADH oxidation

by cysticerci mitochondria was biphasic (Fig. 2A), exhibit-

ing a high-affinity component with a Km app=1.5 AM, a

value within the range reported for typical cytochrome c

a in the presence of different respiratory substrates and inhibitors

Ascorbate+TMPD

ivitya H2O2 productionb Specific activitya H2O2 production

b

0.0 119T9 (4) 0.0

ND ND ND

0.44T0.1 (5) ND ND

0.33c 0.0 ND

TMPD. anat-g O2 min�1 mg�1 protein. bnmol min�1 mg�1 protein. ND,

erage from two determinations.

100

60

20

0 2 4 6 8 10 12

ROTENONE (µM)

ANTIMYCIN (µM)0 2 4 6 8 10 12

100

60

20

100

60

20

20151050

100

A

B

C

CYANIDE (µM)

RAT

E O

F O

XY

GE

N C

ON

SU

MP

TIO

N (

%)

CYANIDE (µM)

0 100 200 300 400 500 600

Fig. 1. Inhibition patterns of cysticerci mitochondrial respiration with

rotenone, antimycin A or cyanide stimulated by exogenous substrates.

Mitochondrial suspensions (1 mg protein/1.8 ml) were preincubated for 3

min at 30 -C with the indicated inhibitor concentrations. Respiration was

started with 1 mM NADH (g), 30 mM succinate (D), or 10 mM ascorbate

plus 150 AM TMPD (?).

I.P. del Arenal et al. / Parasitology International 54 (2005) 185–193188

oxidases [30]. The second component showed much lower

O2 affinity with a Km app around 120 AM. This affinity can

hardly be explained by the existence or participation of a

cytochrome oxidase-type enzyme. In the presence of

cyanide, the high-affinity component was abolished and

unexpectedly the low-affinity curve was decomposed into

two components with apparent Km values of 26 and 92 AM(Fig. 2B). The nature of these two components is discussed

below (in Section 3.3). It should be noted that, not

withstanding the overestimation of O2 concentration by

the Clark electrode, the observed differences in the Km

values are reliable.

In contrast, respiration stimulated by succinate showed a

different pattern, i.e., the O2 saturation curve was mono-

phasic with a Km app for O2=3.3 AM (Fig. 2C). This agrees

with data showing that low cyanide concentrations abol-

ished the succinate-dependent respiration (Fig. 1C).

Cytochrome c is a loosely bound membrane protein

that is partially lost during preparation of mitochondria

[10,31]. Using the cytochrome concentration ratios for c/b

and aa3/b of mammalian mitochondria as reference (e.g.,

1.5 and 2.1, respectively) [32], the concentration ratios

calculated for the same pairs in cysticerci mitochondria

(e.g., 0.6 and 0.2, respectively) indicate that cytochrome

c, as well as cytochrome aa3, is significantly diminished

in cysticerci mitochondria [17,33]. Addition of horse heart

cytochrome c to the mitochondrial preparation induced a

2–3-fold increase in the rate of the NADH-dependent

electron transport (Fig. 2A). This increase can be

attributed to the classical pathway since, under these

conditions, oxygen uptake was completely inhibited by

cyanide. Moreover, the contribution of the low-O2-affinity

pathway to the total respiratory rate became negligible

(Fig. 2A). On the other hand, horse heart cytochrome c

did not cause significant changes in the respiration with

succinate (Fig. 2C).

The Km for O2 of several type aa3 oxidases in

bacteria, parasites and other microorganisms ranges from

0.3 to 3.0 AM [30,34]. Therefore, it is plausible that the

high-O2-affinity (Fig. 2A) and cyanide-sensitive compo-

nent found in the cysticerci mitochondria (Fig. 2B) was

indeed cytochrome aa3 [17]. However, cyanide-resistant

respiration in many parasites has been ascribed to a

putative cytochrome o oxidase [10,13,14]. As shown by

Puustinen and Wikstrom [35], bona fide cytochrome o

contains haem-O. HPLC techniques did not allow

identifying the presence of a cytochrome o (or any other

cytochrome-type oxidase) in cysticerci mitochondria that

could account for the cyanide-resistant pathway [17].

These results were corroborated by photodissociation and

ligand exchange techniques at subzero temperatures, in

which cytochrome aa3 was the only detected oxidase

terminal [17].

3.2. On the nature of the alternative pathway

To get insight into the nature of the alternative

respiratory pathway, classic respiratory inhibitors, as well

as inhibitors of alternative respiratory pathways, were

tested on NADH oxidase activity. All the inhibitors

tested, 50 AM dicumarol or 10 AM rhein (site I), 10

AM 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) or

10 AM myxothiazol (site II), and 100 AM sodium

sulphide or 100 AM azide (site III), induced partial

inhibition (between 10–40%); this inhibition was not

added to that induced by cyanide, antimycin or rotenone.

Compounds such as 150 AM salicylhydroxamic acid

(SHAM), diphenylamine (DPA, 500 AM) and disulfiram

(5.0 AM), all recognized as inhibitors of alternative

pathways [36,37], did not cause additional inhibition of

respiration in mitochondria previously incubated with

cyanide (results not shown). SHAM has also been

reported as an inhibitor of trypanosome alternate oxidase

68 nat-g 02

1.0 min

Aa

b

c

d B

e

f

Fig. 3. Comparative production of hydrogen peroxide by the mitochondrial

respiratory electron chain transport of T. crassiceps cysticerci (A).

Mitochondrial samples (1 mg protein) in 1.8 ml of Hepes 10 mM plus

250 mM sucrose and 0.5 mM EDTA, pH 7.4, were preincubated for 1 min

at 30 -C and respiration was initiated with 5.0 mM NADH (bar) final

concentration. Bovine catalase (60 units) was added at the times indicated

by arrows (traces a–e). Trace f is a cysticerci preparation without catalase.

As a control (B) oxygen uptake of rat liver mitochondria (1.0 mg protein)

were incubated under the same conditions as in A.

120

10080

6040

20

0

140

160180

0 1 2 3 4 5

60

50

40

30

20

100

70

80

0 1 2 3 4 5Vo

(ng

atom

s ox

ygen

min

-1 m

g pr

otei

n-1 )

60

50

40

30

20

10

0

70

80

0.0 0.5 1.0 2.01.5 2.5 3.0

A

B

C

Vo / [02]µM

Fig. 2. Eadie–Hofstee plots of the respiratory O2 desaturation kinetics of T.

crassiceps cysticerci mitochondria in the presence of 5 mM NADH (A), 5

mM NADH plus 1 mM KCN (B) and 30 mM succinate (C). Traces in the

presence of 150 AM horse heart cytochrome c in addition to NADH (A

upper trace) or succinate (C upper trace) are also shown. Respiratory

velocity and O2 concentration were calculated at points selected along the

O2 uptake traces and calculated data used to construct the Eadie–Hofstee

plots were shown.

I.P. del Arenal et al. / Parasitology International 54 (2005) 185–193 189

(TAO), a mitochondrial enzyme insensitive to cyanide

[38]. Quinacrine, at concentrations up to 100 AM, inhibits

flavoproteins that react directly with oxygen [39]. In our

study, 20 AM quinacrine caused 30% inhibition of NADH

and succinate oxidase activities of cysticerci mitochondria,

but this inhibition was not added to that previously

induced by cyanide, antimycin A or rotenone. The overall

results show that cyanide-resistant respiration in cysticerci

mitochondria was resistant to a variety of respiratory

inhibitors, including those reported to cause inhibition of

alternative pathways.

3.3. Hydrogen peroxide production during cysticerci

respiration

The low O2 affinity and the resistance to the wide

array of respiratory inhibitors tested suggest that the

apparent alternative respiratory pathway in cysticerci

mitochondria stems from a spurious reaction of one or

more components of the respiratory chain with O2.

Therefore, we hypothesized that such a reaction could

generate H2O2 as a result of the partial O2 reduction; in

this condition, H2O2 production might be reverted by

catalase. The addition of bovine catalase at different

times during the course of respiration showed that H2O2

was produced and accumulated in significant amounts

with NADH as substrate (Fig. 3A). Under similar

experimental conditions, but using rat liver mitochondria,

no H2O2 production was detected (Fig. 3B). The

production of H2O2 associated to respiration was corro-

borated by the peroxide-dependent formation of ferrithio-

cyanate associated only to NADH as described by

Thurman et al. [27].

The production of H2O2 by cysticerci mitochondria was

measured with different substrates and in the presence of

respiratory inhibitors (Table 1). In the presence of NADH

as electron donor, production of significant quantities of

H2O2 (10% of the total O2 uptake) was detected; this

activity was almost abolished by rotenone and discretely

decreased by antimycin or CN�. This result suggests

complex I as the most likely site for the H2O2 production.

This is in consonance with previous reports showing that

complex I is an important site for H2O2 production in the

respiratory chain of diverse species and tissues of higher

organisms [40,41].

On the other hand, with succinate or ascorbate–TMPD

as electron donor, no H2O2 production was detected with

the techniques used. However, with succinate as electron

donor and in the presence of antimycin or CN�, H2O2

was produced (Table 1) as previously found in mamma-

lian mitochondria [40,42]. This last observation demon-

10

8

6

4

2

0

Vo/[02]µM0 0.1 0.2 0.3 0.4 0.5V

o (n

g at

oms

oxyg

en m

in-1

)

Fig. 5. Eadie –Hofstee plot of the O2-desaturation kinetics of the

endogenous substrate-dependent respiration of whole T. crassiceps

cysticerci. Intact cysticerci (79 mg dry weight) in 1.8 ml of PBS were

allowed to breathe by consuming their endogenous substrates up to

anaerobiosis. Data for the Eadie–Hofstee plot were calculated as in Fig. 2.

70

60

50

40

30

20

10

00.2 0.4 0.6 0.8 1.0 1.2 1.4

Vo[02]µM

NADH + catalase

1.0 min

68 nat-g O2

A

B

Vo

(ng

atom

s ox

ygen

min

-1 m

g pr

otei

n-1 )

Fig. 4. Effect of exogenous catalase on the NADH-dependent oxygen

uptake of cysticerci mitochondria. (A) O2 uptake trace recorded up to

anaerobiosis as evoked by 5 mM NADH in the presence of 60 units of

bovine catalase. (B) Eadie–Hofstee plot calculated from the above trace.

I.P. del Arenal et al. / Parasitology International 54 (2005) 185–193190

strates that antimycin stimulates H2O2 formation during

succinate-supported respiration. The respiratory electron

transport chain of higher organisms normally produces

H2O2 in the range of picomoles per minute per milligram

protein (pmol min�1 mg�1 protein [43–45]. Remarkably,

NADH-dependent respiration in cysticerci mitochondria

produced nanomol quantities per minute milligram protein

of H2O2 (Table 1). Similar amounts of H2O2 production

have been reported in mitochondria of Euglena gracilis

[46].

To establish whether H2O2 was the product of the low-

O2-affinity pathway, the O2 saturation kinetics of NADH-

supported respiration (similar to that shown in Fig. 2A)

was determined in the presence of bovine catalase (Fig.

4). The low-affinity kinetic component for oxygen (with a

Km app=120 AM, Fig. 2A) was not detected; instead, the

kinetic profile showed only the single high-affinity

component (Km app=1.17 AM, Fig. 4). This result

indicates that H2O2 is the spurious product of the low-

affinity pathway.

It was shown (Fig. 2B) that in the presence of NADH

and CN�, the high-affinity component is abolished;

however, the oxygen saturation kinetics in the presence

of CN� was resolved in two low-O2-affinity components

with Km app of 92 and 26 AM. Both disappeared in the

presence of catalase indicating that they accounted for the

H2O2 production (Fig. 4). The first component is very

likely related to that observed in the absence of cyanide

(e.g., Km app around 120 AM); the other could be a second

site of the respiratory chain that leaks electrons when

cyanide blocks complex IV. The identity of the second

component is unknown; however, it has been reported that

mitochondria produce oxygen radicals at the sites of

complexes I and III [40,41].

It has been proposed that H2O2 is the product of an

alternative quinol oxidase or an alternative cytochrome c

oxidase in some helminths [6,10,13]. Our results indicate

that complex I is the major site for H2O2 production in T.

crassiceps cysticerci. Additional sites of the respiratory

chain producing relevant amounts of H2O2 can be

neglected on the following grounds: (i) Previous studies

[17] on haem-composition through HPLC techniques, the

spectroscopic determination of cytochrome–CO adducts

and their corresponding CO/O2 exchange kinetics upon

photolysis at subzero temperatures did not show evidence

for additional oxidases other than cytochrome aa3. (ii)

Complex II oxidizing succinate does not produce appreci-

able amounts of H2O2 (Table 1). (iii) The oxidation of

ascorbate–TMPD by complex IV is fully inhibited by

cyanide (Fig. 1C).

3.4. Oxygen uptake and H2O2 production in whole cysticerci

In the light of the present data, it was important to

ascertain whether H2O2 production by cysticerci mitochon-

dria could be detected in the whole organism. Preliminary

experiments indicated that cysticerci contain enough energy

reserves to maintain endogenous respiration for 24 h or

more. Therefore, the kinetics of O2 uptake was determined

in whole cysticerci utilizing the endogenous substrate

respiration (Fig. 5). Again, two kinetic components were

detected. It is noteworthy that the calculated O2 affinities

(123 and 4.0 AM) in the whole organism (Fig. 5) were close

Fig. 6. Analysis through confocal microscopy of cysticerci (A) stained in green with dihydrorhodamine 123 (57.8 AM�10 min) and in red with MitoTracker

red CMXros (190 nM�10 min) to label mitochondria. An image showing co-localization of the two dyes by means of the combined signal in yellow, due to

overlapping of the green and red signals. Magnification is �60. (B) Transmission electron micrograph of a cysticerci specimen, where numerous mitochondria

(arrow) are shown in the syncytial layer. Bar=1.0 Am. (For interpretation of the references to colour in this figure legend, the reader is referred to the web

version of this article.)

I.P. del Arenal et al. / Parasitology International 54 (2005) 185–193 191

to those found for the NADH respiration in isolated

mitochondria (Fig. 2A).

Production of H2O2 in cysticerci tissues was detected

by incubating the larvae with dihydrorhodamine 123

(DHR). The image visualized by confocal microscopy

(Fig. 6) showed green fluorescence bodies that could be

associated with cysticerci mitochondria. Therefore, it is

probable that H2O2 produced within the mitochondria

reacts with DHR to form fluorescent rhodamine 123. The

localization of the fluorescent bodies in mitochondria was

confirmed by the red-stained image produced after the

addition of the mitochondrion-selective dye, MitoTraker

(MT) [47], to the same preparation (Fig. 6).

In the absence of respiratory organs or a circulatory

system and oxygen carrier proteins, supply of oxygen to

tissues of cestodes depends on diffusion; in this regard, it

has been suggested that an oxygen gradient is established

and that in consequence aerobic metabolism is mainly

limited to the outer layer [14,17]. Accordingly, the DHR-

and MT-treated samples (Fig. 6A) showed that fluores-

cence is associated to mitochondria that are close to the

surface of the cysticerci (syncytial layer). As reference, a

transmission electron micrograph of a cysticercus is also

shown (Fig. 6B). These results indicate that H2O2 is also

produced by the whole cysticerci consuming endogenous

substrates.

3.5. Antioxidant enzymes in cysticerci

Accumulation of H2O2 in cysticerci mitochondria could

be related to limited activity levels of endogenous

enzymes that remove H2O2. Catalase specific activities

of 376 and 15 nmol of O2 min�1 mg�1 protein were,

respectively, detected in cysticerci homogenates and the

mitochondrial fraction. Glutathione peroxidase specific

activities were 252 and 158 nmol of NADP min�1

mg�1 protein for the homogenate and mitochondria,

respectively. Likewise, no evidence was found on gluta-

thione and thioredoxin reductase activities in T. crassiceps

cysticerci, although we recently reported the purification

of a thioredoxin glutathione reductase from this organism

[48]. This thioredoxin glutathione reductase enzyme could

be important to maintain the redox homeostasis in T.

crassiceps cysticerci and is also present in other platy-

helminths [49]. As part of the thioredoxin system,

thioredoxin peroxidase (peroxyredoxines) has been dem-

onstrated to be induced when the organisms are exposed

to oxygen radicals [50,51]. The role of this enzyme system

in the elimination of H2O2 in T. crassiceps cysticerci is

under study.

Considering the low-O2 affinity of the H2O2-producing

pathway (Km app=120 AM, Fig. 2A), the spurious reaction

associated with complex I would require high oxygen

tensions. Thus, one might ask whether the H2O2-producing

pathway would operate at significant rates under the

prevailing pO2 of the parasitic habitat of cysticerci and in

the adult stages of T. crassiceps. pO2 values of 5 and 20

Torr are typical for the intestine and the muscle capillary

network, respectively. These values are 20- and 5-folds

lower than the pO2 found in lung alveoli. Therefore,

production of H2O2 by the adult stage in the nearly

anaerobic environment of the host intestine (5 Torr) seems

I.P. del Arenal et al. / Parasitology International 54 (2005) 185–193192

to be unlikely. On the other hand, limited amounts of H2O2

might be produced by the cysticerci stage in muscle or

brain habitats.

It could be considered that production and excretion of

H2O2 might be part of the strategy used by cysticerci in host

colonization. However, with the techniques used here, we

did not detect H2O2 excreted by the whole cysticerci.

Therefore, the H2O2 produced by mitochondria might be

consumed endogenously.

Acknowledgements

This work was supported by DGAPA-UNAM grants IN-

212600 and IN-236002. We thank R. Moreno-Sanchez, J.P.

Pardo, M. Calcagno Montans and A. Gomez-Puyou for

kindly reviewing the manuscript.

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