Redox status of dogs with non-hodgkin lymphomas. An ESR study

Peter Vajdovicha,*, Tamas Kriskab, Miklos Mezesc, Piroska Ribiczey Szaboa,Nandor Balogha, Andras Banfid, Attila Arany-Tothe, Tibor Gaala, Judit Jakusb

aDepartment of Internal Medicine and Clinics, Faculty of Veterinary Sciences, Szent Istvan University, Budapest, HungarybBio-oxidation Group, Institute of Chemistry, Chemical Research Center, Hungarian Academy of Sciences, Budapest, Hungary

cDepartment of Nutrition, Faculty of Agriculture and Environmental Sciences, Szent Istvan University, Godollo, HungarydPrim-A-Vet Animal Hospital Ltd, Budapest, Hungary

eDepartment and Clinics of Surgery and Ophthalmology, Faculty of Veterinary Sciences, Szent Istvan University, Budapest, Hungary

Received 5 May 2004; received in revised form 16 November 2004; accepted 22 November 2004

Abstract

Free radical and antioxidant parameters in healthy dogs (nZ10) and dogs with non-Hodgkin lymphomas (nZ11) were

measured in blood and lymph node tissue samples before chemotherapy. Enzymatic and other biochemical measurements were

performed. We found that (i) free radical concentrations based on ESR spectra of tissues correlated with higher proliferative

character; (ii) lymphoma cases showed an impaired antioxidant status; (iii) tumors with low oxidative burst capacity and higher

reduced/oxidized glutathione ratio responded better to chemotherapy; and (iv) affected blood and lymph nodes were under

strong oxidative stress.

q 2004 Elsevier Ireland Ltd. All rights reserved.

Keywords: Free radicals; Antioxidant; Canine lymphoma; MDR; Prognosis; Oxidative burst

1. Introduction

A threshold-concept for cancer therapy has been

formulated by Kong et al. [1], stating that low doses of

free radicals may stimulate tumor growth until high

levels of the reactive species or the reduction in

antioxidant activity results in cell death. Lymphomas

comprise one of the most common groups of tumors in

dogs and represent a good model for this quite

0304-3835/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights re

doi:10.1016/j.canlet.2004.11.037

* Corresponding author. Tel.: C361 4784 269; fax: C361 4784

129.

E-mail address: pvajdo@univet.hu (P. Vajdovich).

common disease in humans, as in dogs they are 80%

similar to human medium- and high-grade non-

Hodgkin type lymphomas [2–4]. Several studies

support the idea that increased antioxidant capacity

of lymphomas plays an anti-apoptotic role, therefore

multidrug resistance (MDR) can be developed.

Intracellular thiols, such as reduced glutathione

(GSH), appear to be of special importance in

mediating MDR [5–7]. As in many other neoplastic

diseases, in which chemotherapy or radiotherapy is

commonly used, establishment of a correct treatment

and prognosis is of main importance. Prognosis of

canine lymphomas can be estimated based on several

factors [4,8,9], yet it is not known if alterations of

Cancer Letters 224 (2005) 339–346

www.elsevier.com/locate/canlet

served.

P. Vajdovich et al. / Cancer Letters 224 (2005) 339–346340

the antioxidant character of tumor cells are connected

to the outcome of the treatment. Almost all cancers

examined to date had some imbalance in antioxidant

enzyme levels compared with the cells of origin

[10], but no data are available on the disturbances in

the antioxidant parameters measured in the

circulation of the same tumor-bearing patients. In

addition, clinical studies show contradicting results

on the differences between antioxidant parameters

found in the blood of healthy population and patients

with lymphomas [11,12]. Therefore, the primary aim

of our study was to find out whether there is a

correlation between the redox properties of the blood

plasma, red cells, tumor tissues of dogs with

lymphomas and the clinical outcome of the disease

during therapy. We also compared the levels of these

parameters measured in the blood and lymph node

tissues of healthy animals with those of dogs with

lymphomas.

2. Materials and methods

2.1. Animals

Ten 1.5 to 2-year old healthy beagles (four males,

six females) housed in kennels and 11 dogs with

generalized multicentric lymphomas, housed indoors

or outdoors at the owners’ residence, were entered into

the study. Among affected dogs seven were males and

four females, their ages ranged between 3 and 8 years.

After clinical examination, including abdominal and

cardiac ultrasonography, chest X-ray, routine urina-

lyses, hematological and routine plasma bio-

chemical analyses were performed. Then, the left

prescapular lymph node was surgically excised under

general anesthesia induced by propofol (Diprivan 1%

injection, AstraZeneca, Cheshire, UK) in 4–6 mg/kg

body weight dose, and continued using 1.5–5.5 v/v %

isoflurane (Forane solution, Abbott Labs, Abbott Park,

IL, USA). Staging was established by the guidelines of

World Health Organization’s Clinical Staging System

for Lymphosarcoma in Domestic Animals [3].

2.2. Treatment

Treatment of lymphoma patients was based on a

modified COPA protocol [13] containing the most

frequently used chemotherapeutical drugs for treating

lymphomas: cyclophosphamide (Cytoxan injection,

Bristol–Myers Squibb Co., NY, USA), vincristine

(Vincristine liquid injection, Gedeon Richter Co.,

Budapest, Hungary), prednisolone (Prednisolone

tablet, Gedeon Richter Co., Budapest, Hungary), and

adrimycin (Adrimycin RDFw/PFSw injection, Phar-

macia and Upjohn S.p.A. Co., Milan, Italy). We used

the protocol in 9-week cycles repeating them three

times, then the treatment was stopped and repeated if

the disease relapsed.

2.3. Histoptahology and immunophenotyping

For histopathology, lymph node samples were

examined using paraffin-embedded samples. Routine

hematoxylin–eosin staining was used to classify

and grade the samples according to the REAL

Scheme applied for canine lymphoma [14]. For

immunophenotyping [9], CD3 antibody was used for

T cell, and CD79a for B cell labeling (both from

DAKO Ltd, Cambridgeshire, UK), as well as

Ki67 mouse anti-MIB-1 antibody (Immunotech

Manufacture, Marseille, France) to determine the

proliferative index.

2.4. Sample preparation

All samples were obtained prior to initiation of

chemotherapy. All chemicals were obtained from

Sigma–Aldrich Chemie GmbH (Taufkirchen,

Germany) unless otherwise indicated. Plasma samples

and red blood cell (RBC) hemolysates were prepared

and stored at K20 8C. For plasma vitamin C analysis,

a 50 ml-blood plasma aliquot was added to 150 ml of

sample buffer (5 mM HPO3, 5 mM Na-EDTA),

centrifuged at 1000 g for 10 min at 4 8C. The

supernatant was then stored at K20 8C until further

analysis. For lymph node vitamin C analysis, part of

the lymph node samples was deproteinized by

addition of 3 ml ice-cold 5% trichloro acetic acid

solution to 0.5-g lymph node tissue. Homogenates

were prepared manually in Potter–Elvehjem tubes.

Samples were centrifuged at 5000 rpm, the upper

layer separated, then stored at K20 8C. Lymph node

samples weighing 0.5 g were homogenized manually

in 4.5 ml of Dulbecco ‘A’ phosphate buffered saline

solution using Potter–Elvehjem tubes. Samples were

P. Vajdovich et al. / Cancer Letters 224 (2005) 339–346 341

centrifuged at 5000 rpm, upper layers separated, then

stored at K20 8C. Cells were prepared for ‘oxidative

burst’ assays by placing the extracted lymph nodes

into RPMI 1640 medium containing 10% fetal bovine

serum at 4 8C. The outer membrane was removed and

the tissue passed through a sterile stainless steel sieve.

After sedimentation, single cells were counted and

viability tests performed using trypan blue staining.

2.5. Measurement of free radical and antioxidant

parameters

Reduced (GSH) and oxidized (GSSG) glutathione

concentrations were measured in all samples based on

the original methods of Kosower et al. [15], while the

total antioxidant status was assessed based on ferric

reducing ability of plasma (FRAP), as described by

Benzie et al. [16] Vitamin C [17] and E [18]

concentrations were measured only in lymph node

homogenates and blood plasma. Determination of

lymph node and RBC hemolysate glutathione peroxi-

dase (GSH-Px) and superoxide dismutase (SOD) [19]

activities was accomplished using a Ransel Assay kit

(Randox Laboratories Ltd, Ireland). Thiobarbituric

acid reactive substances (TBARS) expressed as

malondyaldehyde (MDA) were measured in lymph

node homogenates and RBC hemolysates [15]. The

steady state free radical concentration of the

lymphatic tissue was determined using an X-band

electron spin resonance spectrometer (ESR)

constructed by Magnettech GmbH (Berlin, Germany)

[20] The amount of spin adduct was calibrated using

1–100 mM aqueous solutions of a stable nitroxide,

2,2,6,6-tetramethyl-1 piperidinyloxyl (TEMPO;

Fluka, Buchs SG, Switzerland) radical as a standard.

Phorbol myristate acetate (PMA)-induced free radical

production of the lymph node cells was analyzed by

chemiluminescence spectroscopy based on the

description of Allen et al. [21].

2.6. Statistical methods

Unpaired Student’s t-test, correlation analysis and

analysis of linear regression were used to determine

the significance of differences between mean values

of the groups using StatisticaTM program package

(Statsoft, Tulsa, Oklahoma, USA). Only differences

associated with P values less than 0.05 were regarded

as statistically significant.

3. Results

3.1. Clinical staging and histopathology

The clinical stage of 11 canine cases with

multicentric lymphomas was: stage III for three dogs,

stage IVa for seven dogs, and stage IVb for one dog.

Routine hematological and plasma

biochemical examinations did not show marked

alterations from the normal ranges or any other organic

disorders in the dogs entered into the study (data not

shown). Histopathology of lymph nodes in control

dogs revealed two normal and four with chronic

lymphadentitis, often associated with eosinophilic

infiltration, and four nodes with reactive or hyperplas-

tic follicles. Percentage of Ki67-positive cells was

35.8–47.5%. In dogs with lymphomas, seven had

follicular center cell lymphomas (one type I, three type

II, three type III), three diffuse large B-cell lympho-

mas, and one Burkitt-like lymphoma. Percentage of

cells positive to Ki67 marker ranged between 50 and

90%. According to CD79- and CD3-immunopheno-

typing, all lymphoma cases were B-cell lymphomas.

3.2. Redox status of dogs

Significant alterations were found only in

SOD activities and GSH concentrations of RBC-

hemolysates (Table 1). Both parameters showed

significantly lower values compared to controls.

SOD activities, GSH concentrations and GSH/GSSG

ratios of neoplastic lymph nodes also showed lower

values compared to those measured in healthy tissues

(Table 2). No changes in GSH, GSSG, MDA, vitamin

C and E concentrations, total antioxidant status based

on FRAP, SOD and GSH-Px activities measured in

blood plasma samples (data not shown) were found

between control dogs and dogs with lymphomas.

3.3. ESR spectroscopy

We used ESR spectroscopy to measure the actual

total steady state concentration of free radicals in

the lymph nodes (Fig. 1). Table 2 shows that

Table 1

Free radical and antioxidant parameters measured in RBC-hemolysates of control healthy dogs and dogs with lymphomas

MDA

(nmol/g protein)

SOD

(U/g protein)

GSH

(mmol/g protein)

GSSG

(mmol/g protein)

GSH/GSSG

(mmol/g protein)

GSH-Px

(U/g protein)

Control nZ10 415.5G81.5 1185.0G174.8 1858.1G196.1 24.6G11.9 118.3G108.2 107.3G32.2

Lymphoma nZ11 375.0G48.0 797.9***G159.7 1406.8***G223.3 18.39G2.5 77.37G13.9 97.54G29.0

Statistical analysis based on Student’s t-test (***P!0.001). Results are expressed as meansGstandard deviations. MDA, malondialdehyde;

SOD, superoxide dismutase; GSH, reduced glutathione; GSSG, oxidized glutathione; GSH-Px, glutathione peroxidase.

P. Vajdovich et al. / Cancer Letters 224 (2005) 339–346342

the concentrations of free radicals in tumors were

significantly higher than in the corresponding healthy

tissues.

3.4. Respiratory burst

Table 2 shows that oxidative burst monitored by

luminol-dependent chemiluminescence of stimulated

cells isolated from lymph nodes was markedly lower

in those that were prepared from tumor tissues.

Lymph node cells of dogs with lymphomas were

hardly inducible by PMA, whereas those obtained

from healthy dogs showed a relatively high oxidative

burst response (Fig. 2).

3.5. Correlation analysis

We have constructed a statistical correlation matrix

to find relationships among the numerous measured

parameters. Results of the analyses are shown in

Table 3. Higher levels of free radicals in tumors

correlated with low GSH/GSSG ratios in plasma and

with low GSH levels in RBC-s, indicating a close

Table 2

Free radical and antioxidant parameters measured in lymph nodes of cont

MDA

(nmol/g protein)

GSH

(mmol/g protein)

GSSG

(mmol/g p

Control nZ10 118.7G37.7 1240.5G563.8 7.52G6.8

Lymphoma nZ11 119.57G110.9 814.9* G291.8 9.98G4.2

SOD U/g

protein

GSH-Px

U/g protein

F

m

Control nZ10 69.0G28.2 8.4G4.4 1

Lymphoma nZ11 14.6** G8.2 10.8G4.3 1

Statistical analysis based on Student’s t-test (*P!0,05, **P!0.01). Mea

GSH, reduced glutathione; GSSG, oxidized glutathione; SOD, superoxide

based total antioxidant status; TFR, total free radical concentration; iFR, P

piperidinyloxyl.

relationship between the redox status of the tumor and

of the whole organism at late stages of the disease. A

correlation was found between the steady state free

radical concentrations of cells derived from lymph

nodes and their Ki67 proliferation index (Fig. 3),

which confirms the theory that cells at advanced

stages of proliferation are under severe oxidative

stress [20,22–24]. Patients having tumors with higher

GSH/GSSG levels and of decreased ability to produce

free radicals lived longer than those whose tumors

were more inducible (Fig. 4).

4. Discussion

Our results on the antioxidant enzyme activities

and antioxidant status measured in plasma and RBC-

hemolysates in dogs with lymphomas (Table 1) show

similarities to the findings of those who analyzed

plasma antioxidants in leukemic humans. We suppose

that more characteristic findings could be obtained

in cases where severe systemic organ failures are

detected [25]. Nevertheless, the differences in

rol healthy dogs and dogs with lymphomas

rotein)

GSH/GSSG Vitamin E

(mg/g protein)

Vitamin C

(mg/g protein)

5 279.2G171.8 29.6G28.3 219.5G34.1

5 96.19** G57.8 34.7G22.0 207.2G64.6

RAP

mol/g protein

TFR nM TEMPO iFR fmol O2K/

minute

7.3G11.4 423G79 499.6G386.9

8.8G8.9 614**G125 11.9**G3.8

ns and Gstandard deviations is indicated. MDA, malondialdehyde;

dismutase; GSH-Px, glutathione peroxidase; FRAP, iron reduction-

MA-induced free radical production; TEMPO, 2,2,6,6-tetramethyl-1

Fig. 1. Example of ESR spectra taken at 77 K showing the (A) steady-state native free radical concentration of a healthy, and (B) neoplastic

lymph nodes. Arrows show the signal of the Mn/MnO internal standard.

P. Vajdovich et al. / Cancer Letters 224 (2005) 339–346 343

measuerd RBC-GSH and SOD or lymph node-GSH/

GSSG, SOD, TFR and iFR values between control

and lymphoma-bearing groups were so relevant, that

they could not be solely attributed to other factors than

the disease itself. Age and sex of dogs had minor or

contradicting effects on these parameters, while no

Fig. 2. Example of a time course of luminol-dependent chemiluminescen

(control) and tumor-bearing (lymphoma) dogs.

breed-related differences were found in the incidence

of lymphomas among the incoming patients examined

during the time period of our study (data not shown).

The fact that the healthy and affected canine groups

were not exactly age-, sex- and breed-matched does

not negate the fundamental findings of the study.

ce of PMA stimulated cells prepared from lymph nodes of healthy

Table 3

Significant correlations between different parameters measured

from dogs with lymphomas (P! 0.05, nZ8–11), (except for row 5,

where healthy dogs’ data were included as well, nZ15)

Parameters Correlation

coefficient (R)

TFRlymph node vs. GSH/GSSGplasma K0.718

TFRlymph node vs. GSSGplasma 0.729

TFRlymph node vs. GSHRBC-hem K0.819

TFRlymph node vs. Ki-67%lymph node (all dogs) 0.661

Overall survival time vs. Disease free period 0.983

Disease free period vs. iFRlymph node K0.846

Disease free period vs. GSH/GSSGlymph node 0.647

TFR, total free radical concentration; GSH, reduced glutathione;

GSSG, oxidized glutathione; iFR, PMA-induced free radical

production.

Fig. 3. Correlation between total steady state free radical

concentrations and proliferative index (Ki67) of lymph node cells

of healthy and lymphoma-bearing dogs. Correlation coefficient

RZ0.661 (P!0,05).

P. Vajdovich et al. / Cancer Letters 224 (2005) 339–346344

The steady state total free radical concentrations

measured by ESR spectroscopy (TFR) in the tumor

tissues were higher, while the concentrations of GSH

and SOD activities were significantly lower than those

in healthy lymph nodes (Table 2, Fig. 1), suggesting

an oxidative stress and a partial dysfunction of the

antioxidant machinery of lymphomas. This theory is

supported by the finding that negative correlation was

found between the antioxidant property of plasma and

red blood cells (GSH, GSH/GSSG) and the TFR of the

neoplastic lymph nodes in patients (Table 3). Our

findings, however, do not support the general theory

that neoplastic lymph nodes have better antioxidant

defense [5] than the healthy tissue, probably because

at this stage of the disease the antioxidant capacity of

cells has been deteriorated. We found a positive

correlation between disease free period (DFP) of dogs

with lymphomas after therapy and the GSH/GSSG

ratio in their tumor tissues measured before

treatment (Table 3, Fig. 4). Several papers suggest

that high tumor GSH levels may confer resistance to

therapy [5,6], but Ribrag et al. [26] obtained different

results. In their case, GSH content was significantly

lower in chemoresistant non-Hodgkin type lymphoma

cells compared to other lymphoid tissues tested.

Discussed data suggest that this question is more

complicated than it could be explained by a single

theory. This is the first report to show that neoplastic

lymph nodes have a significantly higher TFR and

lower GSH levels, as well as SOD activities in vivo

than the lymph nodes of healthy animals. Moreover

TFR measured in tumors correlated well with the

proliferating index (Ki67%) of the neoplastic tissues

(Table 3 and Fig. 3). This supports the recent idea that,

as a consequence of changes of the redox state, a

series of nano-switches are activated on the cellular

‘switchboard’ [27], which move the cells from

proliferation to various stages of differentiation or

into cell death. Our results corroborate the hypothesis

that free radicals and/or GSH levels may control these

nano-switches in lymphomas: the capacity of cells to

detoxify the oxidants produced is exceeded, which

may trigger a series of events that can induce cellular

dysfunction. PMA-induced free radical production, or

oxidative burst (iFR), was extremely low in cells

obtained from lymphatic tissues, suggesting a loss of

tissue functionality (Table 2, Fig. 2). Elevated TFR in

affected tissues may be a consequence of increased

mitochondrial function in the course of cytochrome-

dependent terminal oxidation and/or damaged anti-

oxidant capacity. Oxidative burst, on the other hand,

is induced in immune cells and generates oxidative

agents by a NADPH-dependent oxidase to kill and

phagocytize harmful pathogens or to trigger lympho-

cyte activation. A cause for decreased oxidative burst

or iFR of neoplastic cells may be due to an altered

composition of the membrane or a damage to

NADPH-oxidase activity [28]. The most relevant

relationship was found between the iFR of cells and

the DFP of dogs with lymphomas (Table 3, Fig. 4).

DFP correlated with the survival time (OST) of

treated dogs (Table 3). Results indicate that dogs with

Fig. 4. Correlation between disease free period (DFP), overall

survival time (OST) of dogs with lymphomas and PMA-induced

oxidative burst measured before chemotherapy. RRFPZK0.847

(P!0,01), ROSTZK0.844 (P!0,01).

P. Vajdovich et al. / Cancer Letters 224 (2005) 339–346 345

lymphomas presenting lower oxidative burst capacity

and higher GSH/GSSG values in lymph nodes

responded much better to COPA chemotherapy than

those with higher free radical-producing ability and

lower GSH/GSSG levels. It has been reported that

patients with malignant lymphomas have free radical

hematic levels different from healthy volunteers due

to lower oxidative burst capacity of their leukocytes

[29,30], but no data are available on measurements

performed using cells derived from lymph nodes

affected by lymphomas. Our data presented in Tables

2 and 3 indicate a considerable loss of functionality

and an increased sensitivity to chemotherapeutical

intervention (Fig. 4) of cells isolated from lympho-

mas. Decreased free radical producing function may

cause lower rate of GSH oxidation, therefore higher

GSH/GSSG values correlated with DFP.

Our findings suggest the possibility of establishing

an additional prognostic tool for asessment of COPA

chemotherapeutical intervention in cases when

obtaining tissue biopsies is possible from the affected

lymph node. Summarizing our findings, proliferating

but poorly differentiated neoplastic lymphoid cells are

under a strong oxidative stress in vivo, which not only

affected the tumor tissue, but also the plasma and red

blood cell glutathione metabolism, as well as SOD

activity. There was a decreased oxidative burst

capacity in tumor cells, which together with their

GSH/GSSG ratio could serve as a prognostic

parameter in canine lymphomas.

Acknowledgements

This work was supported by grants from: Hungar-

ian Scientific Research Fund, no. F35246 AG2.

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