PRECLINICAL STUDY

Oxidative stress and hematological profiles of advanced breastcancer patients subjected to paclitaxel or doxorubicinchemotherapy

C. Panis • A. C. S. A. Herrera • V. J. Victorino • F. C. Campos • L. F. Freitas •

T. De Rossi • A. N. Colado Simao • A. L. Cecchini • R. Cecchini

Received: 10 May 2011 / Accepted: 19 July 2011

� Springer Science+Business Media, LLC. 2011

Abstract Several adverse effects of chemotherapy treat-

ments have been described, and most of these effects are

associated with direct interactions between blood cells and

indirect effects generated during the oxidative metabolism

of antineoplastic drugs. In this study we evaluated the

oxidative systemic status and hematological profiles of

breast cancer patients with advanced ductal infiltrative

carcinoma treated with doxorubicin (DOX) or paclitaxel

(PTX) within 1 h after chemotherapy. Blood analyses

included evaluation of hemogram, pro-oxidative markers,

and antioxidant status. The results showed that advanced

breast cancer diseased (AD) patients without previous

chemotherapy presented anemia and high oxidative stress

status characterized by elevated levels of lipid peroxidation

and nitric oxide, and reduced catalase activity when com-

pared with controls. DOX-treated patients exhibited

increased anemia and reduced antioxidant status, which

was revealed by decreases in reduced glutathione levels

and the total antioxidant capacity of plasma; however,

these changes did not lead to further increases in lipid

peroxidation or carbonyl proteins when compared with the

AD group. PTX-treated patients also showed increased

anemia, lactate dehydrogenase leakage, and enhanced lipid

peroxidation. These data reveal for the first time that

patients subjected to chemotherapy with DOX or PTX

present immediate systemic oxidative stress and red blood

cell oxidative injury with anemia development. These

findings provide a new perspective on the systemic redox

state of AD and patients subjected to chemotherapy

regarding oxidative stress enhancement and its possible

involvement in the aggravation of chronic anemia.

Keywords Breast cancer � Chemotherapy � Oxidative

stress � Anemia

Abbreviations

AD Advanced breast cancer patients

DOX Doxorubicin

PTX Paclitaxel

LDH Lactate dehydrogenase

RBC Red blood cells

TNM Tumor node metastasis classification

MCV Mean cellular volume

SOD Superoxide dismutase

GSH Reduced glutathione

TCA Trichloroacetic acid

TRAP Total antioxidant capacity

ABAP 2,20Azobis

RLU Relative light unities

NO Nitric oxide

TBARS Thiobarbituric reactive substances

MDA Malondialdehyde

DNPH Dinitrophenylhydrazine

AUC Area under the curve

LDL Low density lipoprotein

CL Chemiluminescence

V0 Initial velocity of CL reaction

C. Panis � A. C. S. A. Herrera � V. J. Victorino �F. C. Campos � L. F. Freitas � T. De Rossi �A. L. Cecchini � R. Cecchini (&)

Laboratory of Pathophysiology and Free Radicals, Department

of General Pathology—Center of Biological Science, State

University of Londrina, Londrina, PR 86051-990, Brazil

e-mail: cecchini@uel.br

A. N. Colado Simao

University Hospital, Department of Pharmacy, State University

of Londrina, Londrina, PR, Brazil

123

Breast Cancer Res Treat

DOI 10.1007/s10549-011-1693-x

Introduction

Reactive oxygen species (ROS), despite being products of

normal cellular metabolism, are thought to have a sub-

stantial influence on the development and maintenance of

cancer [1]. Several recent studies have shown high ROS

levels in carcinoma cells compared with the surrounding

healthy tissue [2]. Under normal conditions, ROS are

maintained within narrow boundaries by scavenging sys-

tems, such as superoxide dismutases, peroxiredoxins, and

glutathione-related antioxidant defenses. Consequently,

when the amount of ROS exceeds the capacity of the ROS-

scavenging systems, oxidative stress and imbalanced redox

status occur.

Doxorubicin (DOX) and paclitaxel (PTX) are antineo-

plastic agents largely employed in the treatment of several

types of neoplasia, especially breast cancer [3, 4]. Evidence

shows that adverse effects that occur during chemotherapy

treatment results from both direct contact of intravenous

infusion with red blood cells (RBCs) and systemic oxida-

tive effects generated during drug metabolism [5–7].

DOX is an anthracyclin, metabolic reduction of the qui-

none moiety of which results in one-electron transfer to

molecular oxygen, generating molecules that present strong

oxidizing potential, including superoxide anion, hydrogen

peroxide, and hydroxyl radical [8]. Hydroxyl radicals gen-

erated during the DOX redox cycle participate directly in the

lipid peroxidation process because they induce a disturbance

in membrane organization [9] and are potentially involved in

RBC hemolytic lesion development by iron overload

induced during DOX interactions with ferritin [6]. Recent in

vitro studies have shown that PTX induces oxidative stress

while killing breast cancer cells [10, 11, 12], with production

of hydrogen peroxide and formation of DNA oxidative

adducts after 2 h of treatment [7]. In addition, administration

of antioxidant treatments to breast cancer cells led to

impaired susceptibility to PTX [13], suggesting that gener-

ation of oxidative stress is a secondary antineoplastic

mechanism of action of this drug [14].

Few studies have investigated the parameters of redox

and metabolic changes in patients with breast cancer after

chemotherapy infusion [15, 16] and no differential evalu-

ations regarding cancer disease or treatment-derived

alterations have been considered. The scientific literature is

controversial regarding the oxidative parameters of patients

with breast cancer, who are undergoing chemotherapy. It is

known that both tumor cells and chemotherapy can cause

oxidative stress that benefits tumor cells at the expense of

the patient [17]. In this context, the aim of this study was to

evaluate the potential contribution of chemotherapeutic

agents to the generation of reactive species in advanced

breast cancer disease and its effect on hematological pro-

files 1 h before and after chemotherapy infusion.

Methods

Patient selection and study design

A total of 90 women were recruited at the Londrina Cancer

Institute between January 2009 and September 2010, and

the control group comprised 30 healthy women volunteers.

This study was approved by the Research and Ethics

National Council (CAAE 0009.0.268.000-07), all the

practices were approved by the institutional board, and all

the patients gave informed consent. Patients from the

Londrina Cancer Institute, Londrina-Parana, Brazil, were

characterized by the following parameters: age at diagno-

sis, weight, height, comorbidities, tumor node metastasis

(TNM) classification, and hormonal status. Patients were

assigned to three groups: (1) before chemotherapy (AD

advanced disease group, n = 60), composed of women

with a median age of 51.6 years (range, 33–72) with

advanced breast cancer (TNM stages IIIc and IV) sent to

chemotherapy immediately before PTX or DOX infusion;

(2) after DOX infusion (DOX group, n = 30), composed of

women with a median age of 51.35 years (range, 33–72),

who received DOX 60 mg/m2 intravenously for 1 h; and

(3) after PTX infusion (PTX group, n = 30), composed of

women with a median age of 51.06 years (range, 35–63),

who received PTX 175 mg/m2 intravenously for 1 h. All

patients were found to have infiltrating ductal carcinoma

histological type and received the first cycle of chemo-

therapy treatment. The control group was composed of 30

healthy volunteers with a median age of 52.2 years (range

31–74) recruited from the university campus. Exclusion

criteria included history of previous chemotherapy; current

smoking; hepatic, cardiac, or renal dysfunction; obesity;

drug use; hypertension; sedentarism; diabetes; and other

eventual chronic conditions.

Sample collection and hematological profile

determination

Blood was collected during different periods from the

control group and AD patients, but blood was collected

within 1 h after chemotherapy for the PTX and DOX

groups. Blood was collected in sodium EDTA tubes, and

RBC counting and determination of hemoglobin levels,

hematocrit, and mean cellular volume (MCV) were per-

formed (Coulter STKS). In addition, heparinized blood was

collected and centrifuged for 5 min at 14009g at 4�C to

obtain RBCs. RBCs were washed three times with 0.9%

saline solution at 4�C and used for further analysis. Bio-

chemical analysis of plasma was automatically performed

in Dimension RxL� (Dade-Behring-Siemens Corp.) to

determine lactate dehydrogenase (LDH) levels as an indi-

cator of hemolysis. The osmotic resistance of RBCs was

Breast Cancer Res Treat

123

also evaluated by incubating a 1% erythrocyte suspension

in phosphate buffer containing NaCl solution gradients

ranging from 0 to 1% at 37�C for 1 h. After centrifugation

at 3809g for 10 min, hemolysis was determined by reading

the absorbance of samples at 540 nm.

Antioxidant parameter analysis in RBCs

Catalase activity determination

Erythrocytic catalase activity was determined as described

by Aebi [18]. RBCs were diluted 1:80 in distilled water,

and different volumes were incubated in a system con-

taining 1 M TRIS buffer and 200 mM H2O2 solution.

Absorbance disappearance kinetics was monitored in a

spectrophotometer at 240 nm (Shimadzu UV-1650 PC).

The results are expressed in absorbance values per minute

per milliliter of sample (vabs min-1 ml-1).

Superoxide dismutase (SOD) activity determination

Erythrocytes were hemolyzed at a ratio of 1:20 in distilled

water, incubated in 1 M TRIS buffer and pirogalol

(1.2 mg/ml), and auto-oxidation inhibition was measured at

420 nm in spectrophotometer (Shimadzu UV-1650 PC), as

described by Marklund and Marklund [19]. The results are

expressed as SOD unit/nl of erythrocytes.

Reduced glutathione (GSH) levels

RBCs were hemolyzed at a ratio of 1:10 in distilled water

followed by the addition of 1.25 ml of EDTA and 250 ml

of 50% trichloroacetic acid (TCA). After incubation for

15 min at room temperature, samples were centrifuged at

14009g for 15 min, and 1 ml of the supernatant was added

to a fresh tube containing 2 ml of TRIS buffer (0.4 M, pH

8.9). DTNB was added, and the absorbance of the formed

yellow compound was read at 412 nm [20]. The results are

expressed in nmol/l.

Total antioxidant capacity of plasma (TRAP)

TRAP was determined using 2,20azobis (ABAP) as a rad-

ical generator and luminol to amplify photon detection and

light emission using chemiluminescence (CL), according to

the method of Repetto and collaborators [21]. Plasma

samples were diluted 1:50 in glycine buffer (0.1 M, pH

8.6) at 37�C. ABAP solution was obtained by dissolving

54.24 mg in 1 ml of ultrapure distilled water. Soluble E

vitamin (Trolox) was used as the reference antioxidant

(2.5 mg in 5 ml of glycine buffer [0.1 M, pH 8.6] at 37�C),

and luminol solution was used as the reaction amplifier

(3.98 mg in 250 ll of KOH 1 M added to 10 ml of glycine

buffer and diluted 1:10 at the time of the reaction). CL

curves were obtained in a GloMax luminometer (TD 20/20,

Turner Designs), and the results are expressed in nM of

Trolox.

Pro-oxidative parameters

Measurement of RBCs and plasma lipoperoxidation by CL

reaction

These methods were used for analyzing the integrity of

nonenzymatic antioxidant defenses and the levels of lip-

operoxides formed during exposure to DOX and PTX

chemotherapy. An increase in CL levels is related to pre-

vious in vivo oxidative stress, leading to antioxidant

defense consumption and formation of lipoperoxides, with

consequent photon emissions [22, 23]. RBC lipoperoxida-

tion was evaluated by adding 30 ll of packed erythrocytes

to 3 ml of phosphate buffer, and 1 ml of this solution was

diluted in 12.3 ml of the same buffer. 125 ll of plasma

samples was added to 865 ll of phosphate buffer. The

chemiluminescent reaction was initiated by the addition of

tert-butyl (10 ll) at a final concentration of 3 mM, and the

reaction was read in a GloMax luminometer (TD 20/20

Turner Designers). The results are expressed in relative

light units (RLU), and the obtained curve was used as a

qualitative indicator of lipoperoxidation. Quantitative

results were obtained after area under curve integration

using OriginLab 7.5 software.

Measurement of nitrite levels (NO)

Sample nitrite was measured as an estimate of NO levels

and determined as previously described [24], with adap-

tations for human plasma samples. Plasma aliquots (60 ll)

were deproteinized by the addition of 75 mM ZnSO4

(50 ll), centrifuged at 9,5009g for 2 min at 25�C, fol-

lowed by the addition of 55 mM NaOH (70 ll) (Merck)

and centrifugation at 9,5009g for 5 min at 25�C. The

supernatant was recovered and diluted in glycine buffer

solution (45 g/l, pH 9.7, Merck) at a proportion of 5:1.

Cadmium granules (Fluka) were added to a 5 mM solution

in glycine–NaOH buffer (15 g/l, pH 9.7, Merck) for 5 min,

and this solution was subsequently added to the supernatant

for 10 min. Aliquots were recovered, and the same volume

of Griess reagent was added (Reagent I: 50 mg of N-

naphthylethylenediamine in 250 ml of distilled water;

reagent II: 5 g of sulfanilic acid in 500 ml of 3 M HCl,

Sigma). To determine the nitrite concentration of samples,

a calibration curve was prepared by dilution of NaNO2

(Merck). The absorbance was determined at 550 nm in a

microplate reader.

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123

Thiobarbituric acid reactive substances (TBARS) assay

The TBARS assay was utilized to estimate plasmatic

malondialdehyde (MDA) levels [25]. Plasma (400 ll) was

incubated with 100 ll of 1 mM FeCl3, 100 ll of 1 mM

ascorbic acid, 1 ml of 28% trichloroacetic acid, and 1 ml of

1% thiobarbituric acid at 90�C for 15 min. After cooling,

n-butanol (2 ml) was added, and each tube was vortexed

for 40 s and centrifuged at 14009g for 15 min. The

organic phase was read at 535 and 572 nm in a spectro-

photometer (Shimadzu UV-1650PC) and concentrations

were obtained from the difference between these absor-

bances considering the molar extinction coefficient of

MDA at 535 nm. The results are expressed in nmol/l.

Carbonyl protein content

Carbonyl content was measured as an estimate of protein

oxidative injury [26]. Plasma aliquots (200 ll) were taken

in two tubes. Test tubes received 1 ml of 10 mM dinitro-

phenylhydrazine (DNPH), blank tubes received 1 ml of

2.5 M HCL, and all the tubes were incubated for 1 h in an

ice bath. Samples were successively incubated with

1.25 ml of 20 and 10% solutions of trichloroacetic acid in

an ice bath for 20 min, each with a centrifugation step

between incubations (14009g for 15 min). Supernatants

were discarded, and pellets were treated twice with 1 ml of

an ethanol/water solution (1:1). The final precipitates were

dissolved in 1 ml of 6 M guanidine and incubated for 24 h

at 37�C. Carbonyl content was calculated by obtaining the

355–390 nm spectra of DNPH-treated samples, using one

blank tube for each test. The obtained peaks were used for

calculating the carbonyl concentration using a molar

extinction coefficient of 22 M-1 cm-1. Results are

expressed in nmol ml-1 mg-1 total proteins.

Determination of total protein content

Total proteins were measured to express the carbonyl

content results [27]. Plasma samples were diluted 1:2,000

in 0.9% NaCl and reacted with 300 ll of cupric reagent for

10 min. The mixture was subsequently added to 900 ll of

reagent and incubated in a 50�C bath for 10 min. The

absorbance was read at 660 nm, and sample concentrations

were determined (in mg), using bovine serum albumin for

the standard concentration curve.

Free 8-isoprostane F2 levels in plasma

8-Isoprostane F2 plasma levels were quantified with a

competitive immunoenzymatic kit (ELISA, Cayman

Chemical) based on the activity of 8-isoprostane acetyl-

cholinesterase conjugate. After alkaline hydrolysis of

isoprostane esters in plasma samples, supernatants were

added to the microplate reaction and the absorbance of the

product formed from the reaction between thiocholine and

2-nitrobenzoic acid was read at 412 nm. The limit of

detection of the test was 27 pg/ml. All the sample con-

centrations were determined by comparison with a

recombinant standard curve in pg/ml.

Statistical analysis

Measurements were carried out in triplicate sets for sta-

tistical analysis. Comparisons were performed as follows:

control 9 AD group and AD group 9 DOX or PTX group.

All data are expressed as arithmetic means and standard

errors of means. Differences among groups were assessed

by two-way analysis of variance (ANOVA) followed by

Bonferroni’s test as post hoc to calculate lipid peroxidation

curves and Student’s unpaired t test to calculate other

parameters. Differences were considered statistically sig-

nificant when P \ 0.05. All the statistical analyses were

performed using GRAPHPAD PRISM version 5.0

(GRAPHPAD Software).

Results

Hemogram evaluation revealed that AD patients exhibited a

significant reduction in hemoglobin levels (12.07 ± 0.21

g/dl) and hematocrit (36.67 ± 0.64%) when compared

with the hemoglobin (12.78 ± 0.09 g/dl) and hematocrit

(42.41 ± 0.33%) levels in controls (Table 1). DOX patients

showed significantly reduced hemoglobin levels (11.03 ±

0.34 g/dl) and hematocrit (33.73 ± 1.16%) when compared

with the hemoglobin (12.07 ± 0.21 g/dl) and hematocrit

(42.41 ± 0.33%) levels of the AD group, but the RBC

counts were not significantly different among these groups.

Similarly, the PTX group displayed significantly reduced

circulating RBCs (3.89 ± 0.09 cells/mm3), hemoglobin

levels (11.34 ± 0.24 g/dl), and hematocrit (33.94 ±

0.77%) when compared with the AD group. Both chemo-

therapy groups were clearly characterized as anemic com-

pared with cancer patients who did not receive chemotherapy.

In addition, VCM did not vary in any chemotherapy condi-

tion, indicating a normocytic anemia. No variations were

observed in cell fragility and 8-isoprostane levels in all the

evaluated groups, but significantly higher LDH levels were

found in PTX patients (361.7 ± 25.93 U/l) compared with

the AD group (225.1 ± 28.11 U/l). Significantly higher lev-

els of carbonyl content were found in the AD group

(91.1 ± 5.25 nmol/l 9 mg proteins-1) when compared with

controls (77.78 ± 4.33 9 mg proteins-1), whereas DOX

patients showed reduced levels (82.01 ± 3.29 nmol/l 9 mg

proteins-1) when compared with the AD group.

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Lipoperoxidation curves were evaluated using three

statistical parameters (Fig. 1). Two-way ANOVA and

Student’s t test were used for analyzing the difference

among total curve profiles while Bonferroni’s test was used

for comparing between the point from the curves. As

shown, the lipoperoxidation profiles of cancer patients

displayed significant levels of lipoperoxidation when

compared with controls in all the statistical analyses.

Integration of the area under the curve (AUC) did not

reveal significant differences but Bonferroni’s test analysis

showed six significant points of difference between the

CTR and AD groups. Evaluation of DOX patients revealed

a significant increase in the initial rate of lipoperoxidation,

as shown in the ascending part of the curve, but no points

were statistically significant in Bonferroni’s test, Student’s

t test, or AUC. The PTX group displayed significantly

higher levels of lipoperoxidation only when evaluated by

Student’s t test.

Plasma lipoperoxidation curves (Fig. 2) were evaluated

using the same statistical parameters as applied to the RBC

curves. The AD group exhibited highly significant levels of

plasma lipoperoxidation when compared with controls using

all statistical parameters, resulting in 53 points of difference

between the curves. DOX treatment also resulted in elevated

lipoperoxidation levels relative to the AD group, with 34

significant points. Although the PTX group was statistically

different from the AD group, there were no points of dif-

ference in Bonferroni’s test evaluation. The AUC did not

show any significant alteration in the DOX and PTX groups.

Nitrite measurement (21.21 ± 1.78 lM) and TBARS levels

(158.3 ± 16.33 nmol/l) were elevated only in AD patients

when compared with control nitrite (16.47 ± 0.82 lM) and

TBARS (99.88 ± 6.46 nmol/l) levels.

Evaluation of antioxidant defenses (Fig. 3) indicated a

significant decrease in catalase activity in AD patients

(527.6 ± 11.96 vabs min-1 ml-1) in relation to controls

(562.6 ± 9.83 vabs min-1 ml-1) while SOD was not

altered in any of the groups. GSH (12.03 ± 0.88 nmol/l)

and TRAP levels (273.3 ± 20.45 nM) were significantly

reduced only in DOX patients when compared with the

Table 1 Hematological and plasmatic oxidative parameters

Control AD DOX PTX

Hemoglobin (g/dl) 12.78 ± 0.09 12.07 ± 0.21* 11.03 ± 0.34# 11.34 ± 0.24#

Hematocrit (%) 42.41 ± 0.33 36.67 ± 0.64* 33.73 ± 1.16# 33.94 ± 0.77#

RBCs counting (cells/mm3) 4.94 ± 0.09 4.15 ± 0.07 4.03 ± 0.10 3.89 ± 0.09#

Mean corpuscular volume (VCM, l3) 90.83 ± 1.37 88.36 ± 1.33 86.85 ± 1.20 86.91 ± 2.04

LDH (U/l) 201.4 ± 14.23 225.1 ± 28.11 274.2 ± 22.11 361.7 ± 25.93#

8-F2-isoprostanes levels (pg/ml) 144.8 ± 0.27 144.9 ± 0.26 144.3 ± 0.29 144 ± 0.15

Osmotic fragility (area integration) 49 ± 4.34 59 ± 10.1 51.5 ± 8.9 59 ± 9.89

Carbonyl content (nmol/l 9 mg proteins-1) 77.78 ± 4.33 91.1 ± 5.25* 82.01 ± 3.29# 90.12 ± 5.24

* P \ 0.05 when compared to controls and #when compared to AD group

Statistical Analysis CTR X AD AD X DOX AD X PTX

Two-way ANOVA P<0.001 P<0.001 P=0.3497

Bonferroni’s Test P<0.01Points 16-21

No points No points

Curves Student’s t Test P<0.0001unpaired

P=0.1011paired

P<0.0001paired

AUC Student’s t Test P=0.0771unpaired

P=0.5502paired

P=0.7617paired

BA

Fig. 1 Lipid peroxidation of RBCs. Lipid peroxidation of erythro-

cytic membrane was assessed as indicative of oxidative injury.

Lipoperoxidation (a) and statistical significance of the curves (b).

Individual distribution of values and means were evaluated by

Student’s unpaired t test. CRT controls, AD advanced disease group,

DOX breast cancer patients treated with DOX infusion 60 mg/m2/

60 min, PTX breast cancer patients treated with PTX infusion

175 mg/m2/60 min, AUC integration of area under the curve *indi-

cate statistical difference when related to CTR, #compared to AD

group (P \ 0.05)

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123

GSH (17.16 ± 1.56 nmol/l) and TRAP levels (345.8 ±

26.03 nM) of the AD group.

Discussion

Although the mechanism by which DOX and PTX cause

anemia after 1 h of chemotherapy is not understood, evi-

dence shows that systemic oxidative stress accompanies

breast cancer patients regardless of whether they are

undergoing chemotherapy or not.

Hemogram evaluation showed that AD patients had

chronic anemic status and that both chemotherapy treat-

ments enhanced anemia by lowering hemoglobin levels

and hematocrit. In PTX patients, hemoglobin reduction

could be a consequence of decreased circulating RBCs

while it could be related to the possible reaction of

hemoglobin with DOX [28] in the DOX group since we did

not observe altered RBC quantity in this group. Corpus-

cular mean volume suggested that the anemic process

detected in our patients was related to immediate hemolytic

injury because of normocytic normochromic anemia.

A possible mechanism of cellular injury by PTX pro-

posed by other authors is increased production of hydro-

peroxides by PTX, leading to oxidative stress in human

lung cancer cells and breast cancer cells [11, 29]. Fur-

thermore, Alexandre et al. noted a significant induction of

H2O2 release after 1 h of PTX treatment in A549 lung

cancer cells [11], but the relationship of oxidative stress to

the overall mechanism by which PTX causes damage to

cells is not well understood. Further, lipoperoxidation

detection of RBC membranes was possible when using the

high sensitivity CL method, which allows the detection of

very low levels of lipid peroxides pre-formed in vivo. This

method also provides a view of nonenzymatic antioxidant

defenses based on the previous oxidative stress suffered by

cells and resulting in increased photon emission, as we

have previously reported in in vitro models [22, 30].

Experimental evidence of the acute oxidative effects of

DOX has demonstrated that this treatment enhances lipo-

peroxidation rates in rat cardiomyocytes [31] and in human

LDL [32], but this mechanism has not been previously

demonstrated in human RBCs. In this study, the chemo-

therapy treatments did not differ with respect to increased

Statistical Analysis CTR X AD AD X DOX AD X PTX

Two-way ANOVA P<0.001 P<0.001 P=0.3497

Bonferroni’s Test P<0.0001Points 8-60

P<0.0001Points 13-17Points 32-60

P<0.0001No points

Curves Student’s t Test P<0.0001unpaired

P<0.0001paired

P<0.0001paired

AUC Student’s t Test P=0.0010unpaired

P=0.1138paired

P=0.1760paired

B

DC

A

Fig. 2 Plasmatic pro-oxidative parameters. Plasma lipoperoxidation

curves (a), statistical evaluation of lipoperoxidation curves (b), NO

(c), and TBARs levels (d) were evaluated as indicative of oxidative

status. Individual distribution of values and means were evaluated by

Student’s unpaired t test. CTR controls, AD advanced disease group,

DOX breast cancer patients treated with DOX infusion 60 mg/m2/

60 min, PTX breast cancer patients treated with PTX infusion

175 mg/m2/60 min, AUC integration of the area under the curve.

*indicate statistical difference when related to CTR, #compared to AD

group (P \ 0.05)

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123

oxidative stress, but DOX treatment appeared to produce a

different pattern of RBC lipid peroxidation as evidenced by

the slight shift of the curve to the left. CL is a very sensitive

method that takes into account a kinetic analysis of the

ascending part of the emission curve under the assumption

that the variation in V0 (initial velocity) values depends on

the level of pre-existing lipid peroxide in the tissue and

membrane disarrangement [33, 34]. This information is

useful when considering the dislocation of the CL curve to

the left in the groups studied. Peres and collaborators [46]

have previously shown that when epidermis cells are

gradually subjected to lipoperoxidation, the dislocation of

the curve to the left occurs according to the time that skin

homogenates remain exposed to oxidation. The displace-

ment to the left of the CL chart obtained by skin oxidation

represents modification of the lipid structure of the cell

membrane. The exposure or destruction of lipids in the cell

layer facilitates propagation of the chain reaction [34].

The high levels of plasma CL observed in cancer

patients were exacerbated by PTX and reduced by DOX

treatments. PTX reportedly induces oxidative stress in

cancer cells [11, 12]. However, the initially increased

TBARS levels in AD patients did not show any additional

changes after chemotherapy treatment. The same profile

was observed for NO levels. NO is a pleiotropic regulator

of many physiological processes and may have dual pro-

and anti-tumor effects [35–38]. Abdelmagid and Too [39]

showed that intracellular production of NO in the human

MCF-7 breast cancer cell line increases cell viability and

inhibits cell apoptosis. Experimental studies have reported

increases in TBARS and NO after DOX/PTX acute infu-

sion [40]. The results of this study showing reduced CL in

patients subjected to DOX when compared with AD

patients are supported by the reduced carbonyl compounds

found.

Analysis of antioxidant parameters revealed decreases in

GSH and TRAP in DOX-treated patients when compared

with advanced cancer patients. GSH is a hydrophilic

intracellular molecule that participates in conjugation

reactions during phase II of xenobiotic metabolism. In vitro

studies have demonstrated that the transport of DOX to the

outside of RBCs occurs via RLIP76, an ATP-dependent

transporter of glutathione conjugates, which participates in

the regulation of lipoperoxidation metabolites during oxi-

dative stress induced by xenobiotics [41]. Furthermore,

DOX itself stimulates the hexose monophosphate shunt,

leading to glutathione oxidation and GSH requisition [42].

Thus, the reduction of GSH and TRAP observed in DOX

patients could be related to oxidative consumption of thiol

residues and low molecular weight antioxidants by drug

metabolism, without the involvement of antioxidant enzy-

matic defenses, whereas PTX did not compromise antiox-

idant defenses.

Chemical data regarding in vivo pharmacokinetics

support our hypothesis of immediate DOX effects, indi-

cating that it has a rapid distribution half-life (5–10 min)

and its first biotransformation occurs approximately 12 min

after infusion [43], which could contribute to the

Fig. 3 Antioxidant parameters.

Catalase (a), SOD (b), GSH

content (c), and TRAP levels

(d) were evaluated as

antioxidant parameters.

Individual distribution of values

and means were evaluated by

Student’s unpaired t test. CRTcontrols, AD advanced disease

group, DOX breast cancer

patients treated with DOX

infusion 60 mg/m2/60 min, PTXbreast cancer patients treated

with PTX infusion 175 mg/m2/

60 min. *indicate statistical

difference when related to CTR,#compared to AD group

(P \ 0.05)

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123

immediate effects on circulating RBCs that we observed in

this study. Colombo and collaborators [44] demonstrated

experimentally that 50% of the DOX dose is transported by

RBCs and increases with higher doses, suggesting the

elevated storage capacity of these cells. Thus, this evidence

supports the hypothesis that the early RBC–DOX interac-

tion leads to hemolytic damage, which can result from both

the direct action of DOX on the membrane and ROS-

mediated injury. Regarding the immediate effects of PTX,

a recent study showed that PTX could trigger RBC-pro-

grammed cell death, a process called eryptosis, by

increasing cytosolic calcium and exposing phosphatidyl

serine at the cell surface [45]. The increased LDH in

patients treated with PTX revealed oxidative pre-hemolytic

injury induced by the drug, as observed in vitro previously

[22].

In conclusion, oxidative stress is an ultimate participant

in the harmful systemic processes in advanced cancer

patients, and treatment with chemotherapy drugs sustains

these injuries. The occurrence of immediate anemia in

breast cancer patients after 1 h of chemotherapy adminis-

tration is new information regarding chronic disease ane-

mia. In addition, we have shown that DOX and PTX

displayed an oxidative mechanism that may be involved in

the RBC hemolytic injury pathway.

Acknowledgments The authors thank Jesus Vargas for his excep-

tional technical assistance, and the Fundacao Araucaria, CNPq, and

CAPES for providing financial support.

Conflict of interest The authors declare that they have no com-

peting interests.

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