or Î²-cryptoxanthin at physiological serum

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Article

Effects of plant sterols and/or #-cryptoxanthin at physiological serum concentrations on suicidal erythrocyte death Andrea Alvarez-Sala, Gabriel López-García, Alessandro Attanzio, Luisa Tesoriere, Antonio Cilla, Reyes Barbera, and Amparo Alegria J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05575 • Publication Date (Web): 18 Jan 2018 Downloaded from http://pubs.acs.org on January 18, 2018

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Journal of Agricultural and Food Chemistry

Effects of plant sterols and/or β-cryptoxanthin at physiological serum concentrations on suicidal erythrocyte death

Andrea Alvarez-Salaa§, Gabriel López-Garcíaa§, Alessandro Attanziob*, Luisa Tesoriereb, Antonio Cillaa*, Reyes Barberáa, Amparo Alegríaa §

a

These authors have contributed equally and must be considered as first authors

Nutrition and Food Science Area. Faculty of Pharmacy, University of Valencia, Avda. Vicente Andrés

Estellés s/n, 46100 - Burjassot (Valencia), Spain b

Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF),

University of Palermo, Via Archirafi 28, 90123 - Palermo, Italy

*Corresponding authors: [email protected] (+34963544972) and [email protected] (+3909123896819)

1 ACS Paragon Plus Environment


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1

Abstract

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The eryptotic and hemolytic effects of a phytosterol (PS) mixture (β-sitosterol,

3

campesterol, stigmasterol) and/or β-cryptoxanthin (β-Cx) at physiological serum

4

concentration

5

butylhydroperoxide (tBOOH) (75 and 300 µM) were evaluated. β-Cryptoxanthin

6

produced an increase in eryptotic cells, cell volume, hemolysis and glutathione

7

depletion (GSH), without ROS overproduction and intracellular Ca2+ influx. Co-

8

incubation of both bioactive compounds protected against β-Cx-induced eryptosis.

9

Under tBOOH stress, PS prevented eryptosis, reducing Ca+2 influx, ROS

10

overproduction and GSH depletion at 75 µM, and hemolysis at both tBOOH

11

concentrations. β-Cryptoxanthin showed no cytoprotective effect. Co-incubation with

12

both bioactive compounds completely prevented hemolysis and partially prevented

13

eryptosis, as well as GSH depletion induced by β-Cx plus tBOOH. Phytosterols at

14

physiological serum concentrations help to prevent pro-eryptotic and hemolytic effects,

15

and are promising candidate compounds for ameliorating eryptosis-associated diseases.

16

Keywords: Phytosterol, β-cryptoxanthin, eryptosis, hemolysis, oxidative stress.

and

their

effect

against

oxidative

17


stress

induced

by

tert-

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INTRODUCTION

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Eryptosis is defined as programmed erythrocyte death (similar to apoptosis in nucleated

20

cells), and is characterized by cell shrinkage and membrane blebbing with

21

phosphatidylserine exposure at the erythrocyte surface. Eryptosis is the principal form

22

of erythrocyte death, though there are also other death processes such as hemolysis,

23

with the subsequent release of hemoglobin into the bloodstream.1 Excessive eryptosis is

24

observed in several clinical conditions, including anemia, atherothrombosis,

25

atherosclerosis, thalassemia, hemolytic uremic syndrome, cardiac and renal failure,

26

Wilson’s disease, obesity, diabetes, chronic inflammatory diseases and malignancies, as

27

well as in the elderly. However, eryptosis can also contribute to protect against malaria

28

disease, attenuating parasitemia.2-9

29

Eryptosis is triggered by several stressors such as osmotic shock, energy depletion,

30

hyperthermia and oxidative stress. The latter is a powerful trigger of eryptosis,10-11 and

31

it is assumed that the use of antioxidant compounds may prevent this phenomenon. In

32

vitro studies have demonstrated the anti-apoptotic and antioxidant effects of

33

phytosterols (PS)12-13 and β-cryptoxanthin (β-Cx)14-15 on human colon adenocarcinoma

34

cells. The PS, naturally occurring mainly in vegetable oils, margarines and nuts, are

35

steroids associated with health benefits such as cholesterol-lowering, anti-proliferative

36

and anti-inflammatory effects.16 While β-Cx is a xanthophyll, contained primarily in

37

citrus fruits, related with protective effects against osteoporosis, inflammatory diseases

38

and oxidative stress.17-18 However, their activity in relation to erythrocyte death has not

39

been studied to date. Compounds structurally similar to PS such as whitaferin A at

40

concentrations effective against breast cancer cells (1-10 µM)19 or oxysterols at

41

concentrations occurring in hyper-cholesterolemic subjects (20 µM)20 have shown a

42

pro-eryptotic effect associated to oxidative stress, which could contribute to the 3 ACS Paragon Plus Environment


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development of atherosclerosis and thrombosis. Likewise, dietary compounds

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structurally similar to β-Cx, such as retinoic acid (1-10 µM)21 and fucoxanthin (25-75

45

µM),22 produced pro-eryptotic effects through intracellular Ca2+ influx and

46

phosphatidylserine translocation, without disturbing the redox balance. Although these

47

compounds led to eryptosis, it is important to underscore that the existing chemical

48

structural differences (keto-, epoxy-, hydroxy- and ester groups, among others) with

49

respect to PS and β-Cx could imply different biological effects.

50

A previous study carried out by our group showed that four months of consumption

51

of PS (1.5 g/day)-enriched milk-based fruit beverage added with β-Cx (0.75 mg/day)

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resulted in an increase in the serum concentration of PS (β-sitosterol, campesterol and

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stigmasterol) (18 to 22 µM) and β-Cx (0.3 to 1 µM).23-24 The increase in serum

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concentration was associated to a decrease in osteoporosis and cardiovascular risk

55

(decrease in cholesterolemia), suggesting a synergistic effect of both bioactive

56

compounds.17 With regard to their positive effect on cardiovascular risk, it is possible

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that these bioactive compounds can to help prevent atherosclerosis induced by

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eryptosis, since it has been reported that phosphatidylserine exposure in eryptotic

59

erythrocytes promotes their adhesion to the endothelium, thereby leading to vascular

60

complications.10,25 In this regard, the present study for the first time has evaluated the

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pro- or anti-eryptotic and hemolytic effects of a mixture of PS (β-sitosterol, campesterol

62

and stigmasterol) and/or β-Cx at concentrations obtained after the intake of PS-enriched

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milk-based fruit beverages containing β-Cx, and their cytoprotective effect against

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oxidative stress induced by tert-butyl hydroperoxide (tBOOH), as a novel and hitherto

65

non-described mechanism of action complementary to their known hypocholesterolemic

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and anticancer effects.

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MATERIALS AND METHODS 4 ACS Paragon Plus Environment

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Reagents

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(24S)-Ethylcholest-5,22-dien-3β-ol (stigmasterol) (purity 97%), (24R)-ethylcholest-5-

70

en-3β-ol (β-sitosterol) (purity 97.3%), and Luperox® tBOOH solution were obtained

71

from Sigma Chemical Co. (St. Louis. MO, USA). (24R)-Methylcholest-5-en-3β-ol

72

(campesterol) (purity 96.19%) was purchased from Chengdu Biopurify Phytochemicals,

73

Ltd. (Sichuan, China). β-Cryptoxanthin (purity 97%) was obtained from Cymit Química

74

(Barcelona, Spain).

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Preparation of erythrocyte cultures and treatment conditions

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Blood was drawn in K2EDTA tubes from healthy human volunteers (n = 8; aged 24–59

77

years; normal body mass index) at health service of University of Valencia, with

78

informed consent. Erythrocytes were isolated by centrifugation (1,000 rpm, 4 ºC, 25

79

min) and washed with PBS according to Tesoriere et al.20 The cells (0.4% v/v

80

hematocrit) were incubated for 48 h (37 ºC, 5% CO2, 90% humidity) in the absence or

81

presence of PS and/or β-Cx (alone or in combination) prepared at 0.2% (v/v) ethanol

82

final concentration in Ringer solution containing 125 mM NaCl, 5 mM KCl, 1 mM

83

MgSO4, 32 mM HEPES, 5 mM glucose and 1 mM CaCl2.26 The PS mixture [22 µM PS

84

mix: β-sitosterol (13 µM), campesterol (8 µM) and stigmasterol (1 µM)] and/or β-Cx (1

85

µM) used were compatible with physiological serum concentrations obtained after the

86

consumption of PS-enriched milk-based fruit beverages containing β-Cx, as reported in

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previous studies carried out by our group.23-24 Control cells were incubated with ethanol

88

0.2% (v/v). Oxidative stress was induced with tBOOH at 75 and 300 µM - this range of

89

concentrations being similar to those of previous reports27-28 - during the last 30 min of

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the 48 h incubation period in the presence or absence of bioactive compounds (37 ºC,

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5% CO2, 90% humidity). 5 ACS Paragon Plus Environment


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Measurement of annexin V and forward scatter (FSC)

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The measurement of eryptosis was performed through the externalization of

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phosphatidylserine to the cell surface, according the protocol of the manufacturer

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(annexin V apoptosis detection kit I, BD Biosciences, San Diego, CA, USA). Briefly,

96

cell suspension (30 µL) was added to a new tube and washed with PBS. Then, cells

97

were centrifuged (800 rpm/25 ºC/ 5 min) and suspended in binding buffer (100 µL). To

98

the cell suspension 5 µL of annexin V-FITC was added and incubated at room

99

temperature for 15 min in the dark. Then, 400 µL of PBS was quickly added and the

100

cells were analyzed by FSC and annexin V fluorescence intensity by flow cytometry

101

(FACS Canto, BD Biosciences), evaluating 1 x 104 events for each sample (ʎexc = 488

102

nm and ʎem = 525 nm). The analysis of FSC was displayed as percentage of erythrocytes

103

undergoing a threshold for shrunken (80) erythrocytes. This

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correlation has been described previously by Jemaà et al.26

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Determination of hemolysis

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Erythrocytes were centrifuged (1,800 rpm/ 24 ºC/ 5 min) and the concentration of

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hemoglobin in the supernatant was determined at 405 nm by spectrophotometry (Perkin

108

Elmer, Lambda 2) according to Tesoriere et al.20 The absorbance of the supernatant

109

from analogous erythrocytes lysed in distilled water was defined as 100% hemolysis.

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Measurement of cytosolic Ca2+

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Intracellular Ca2+ concentration was measured according to Tesoriere et al.29 using the

112

cell permeable probe Fluo-3 AM (Santa Cruz Biotechnology, Santa Cruz, CA, USA),

113

whose fluorescence is directly representative of the ion concentration. Briefly, cell

114

suspension (55 µL), PBS (445 µL) and Fluo-3/AM at 2 μM final concentration were

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added to a tube, followed by incubation during 40 min (37 ºC, 5% CO2, 90% humidity).

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After centrifugation (800 rpm, 24 ºC, 5 min), erythrocytes were suspended with 500 µL


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of PBS and analyzed by flow cytometry (FACS Canto, BD Biosciences), evaluating at

118

least 1 x 104 events for each sample (ʎexc = 506 nm and ʎem = 526 nm).

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Measurement of intracellular reactive oxygen species (ROS)

120

Intracellular ROS accumulation was measured by changes in fluorescence resulting

121

from the oxidation of 2´,7´-dicholofluorescein diacetate (DCFDA) (Sigma Chemical

122

Co., St. Louis, MO, USA), as previously reported.20 Cell suspension (55 µL), PBS (445

123

µL) and DCFDA at 10 μM final concentration were added to a tube and incubated

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during 40 min (37 ºC, 5% CO2, 90% humidity). Erythrocytes were collected by

125

centrifugation (800 rpm, 24 ºC, 5 min), suspended in 500 µL PBS and subjected to flow

126

cytometric analysis (FACS Canto, BD Biosciences). At least 1 x 104 cells were

127

analyzed for each sample (ʎexc = 495 nm and ʎem = 529 nm).

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Measurement of intracellular glutathione (GSH)

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The evaluation of cellular GSH content was made with 1 µM (final concentration) of 5-

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chloromethylfluorescein diacetate (Green CMFDA) (Abcam, Cambridge, UK),

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according to Officioso et al.28 The CMFDA was added to a tube with 55 µL cell

132

suspension and PBS (445 µL). Then, erythrocytes were incubated during 40 min (37 ºC,

133

5% CO2, 90% humidity) and collected by centrifugation (800 rpm, 24 °C, 5 min). Cells

134

were suspended in 500 µL PBS and analyzed by flow cytometry (FACS Canto, BD

135

Biosciences), evaluating at least 1 x 104 events for each sample (ʎexc = 492 nm and ʎem

136

= 516 nm).

137

Statistical analysis

138

One-way analysis of variance (ANOVA) followed by the Tukey post-hoc test was

139

applied to determine differences between the different samples. A significance level of

140

p < 0.05 was adopted for all comparisons, and the Statgraphics® Centurion XVI.I

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statistical package (Statpoint Technologies Inc., VA, USA) was used throughout.



142 143

RESULTS AND DISCUSSION Effect of PS and/or β-Cx on eryptosis

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Forward scatter, annexin V binding and cytosolic Ca2+

145

Eryptosis is characterized by cell shrinkage. The effect of the bioactive compounds (PS

146

and/or β-Cx) upon cell volume was therefore evaluated through forward scatter (FSC)

147

detection (see Figure 1). Incubation for 48 h with PS did not produce significant

148

changes (p < 0.05) in cell volume, while treatment with β-Cx increased the geometric

149

mean two-fold versus the control. Co-incubation with PS and β-Cx blunted the effect of

150

β-Cx alone (31% decrease in cell volume), suggesting that PS partially prevent the

151

increase in cell volume (Figure 1 A and B). However, two differentiated erythrocyte

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populations were observed under our experimental conditions: one with low FSC (< 20)

153

(Figure 1 C) and another with high FSC (˃ 80) (Figure 1 D). In the case of PS, the

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erythrocyte population was very similar to the control. β-Cryptoxanthin in turn had a

155

marked effect upon erythrocyte population distribution, decreasing the percentage of

156

cells with low FSC (53%), with a concomitant increase in high FSC cells (143%) versus

157

the control. In agreement with the results referred to β-Cx, erythrocyte exposure during

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48 h to fucoxanthin (25-75 µM)22 produced a subpopulation of erythrocytes exhibiting

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cell shrinkage (FSC < 200), and another with signs of swelling (FSC ˃ 800). However,

160

the proportions of both erythrocyte subpopulations (12-20% and 1.5-3%, respectively)

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reported by the authors differed from those observed with β-Cx, where high FSC cells

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predominated. This behavior may be related to two possible death pathways of the

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erythrocytes. It has been reported that a reduction in cell volume is associated to

164

eryptosis, while a swelling cell is associated to hemolysis.1 Therefore, β-Cx may

165

partially act through hemolytic effects in its erythrocyte death-inducing action. The

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simultaneous presence of both bioactive compounds (PS and β-Cx) significantly

167

reduced the percentage of cells with low FSC (69% versus control); it is therefore 8 ACS Paragon Plus Environment

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suggested that PS blunted (37%) the increase in erythrocytes with high FSC induced by

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β-Cx alone.

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One of the main characteristics of eryptosis is cell membrane scrambling, with the

171

exposure of phosphatidylserine at the external cell surface.30 As illustrated in Figure 2

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A, β-Cx unlike PS induced a pro-eryptotic effect through an increase in the percentage

173

of annexin V-binding erythrocytes (56% with respect to control cells) – this implying

174

the translocation of phosphatidylserine to the external cell membrane surface. The dot

175

plot analysis (FSC versus annexin V) indicate that FSC ˃ 80 cells show translocation of

176

phosphatidylserine after β-Cx treatment (see Figure 2 B), suggesting an eryptotic-

177

hemolytic effect. Nevertheless, co-incubation with both bioactive compounds prevented

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the eryptosis induced by β-Cx, indicating a cytoprotective effect of PS. On the other

179

hand, an increase in intracellular Ca2+ is considered a key event in the eryptotic

180

process,28 though it does not always account for the triggering of cell membrane

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scrambling.19 Figure 3 A shows that none of the treatments with bioactive compounds

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produced an increase in intracellular Ca2+, despite activation of the translocation of

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phosphatidylserine by β-Cx. This fact suggests that eryptosis induction by β-Cx is

184

triggered through Ca+2-independent mechanisms. In agreement with these results, it has

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been reported that some carotenoids such as fucoxanthin (50-75 µM)22 and retinoic acid

186

(1-10 µM)21 trigger phosphatidylserine translocation to the cell surface, though unlike in

187

our study an increase in intracellular Ca2+ was observed. It has been described that the

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activation of protein kinase C31 or caspase-332 triggers membrane phospholipid

189

scrambling in the absence of Ca+2 in healthy human erythrocytes. The authors found no

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clear correlation between elevated calcium levels and phosphatidylserine exposure,

191

suggesting that more molecular pathways are involved.



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In contrast to our results with PS, the incubation of human erythrocytes with steroidal

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compounds such as whitaferin A (5 and 10 µM)19 or oxysterols (20 µM)20 during 48 h

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showed pro-eryptotic effects through an increase in phosphatidylserine translocation (≈

195

2.5-5 and 50-fold, respectively) and intracellular Ca+2 influx (≈ 1.3-1.6 and 20-fold,

196

respectively). The different behavior observed could be due to the absence in PS of

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reactive functional groups based on oxygen, such as keto-, epoxy- and ester groups,

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which have been reported to promote lipid membrane peroxidation and apoptosis in

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endothelial cells.33 Moreover, in vivo studies have demonstrated that PS incorporation

200

into the erythrocyte membrane does not alter membrane properties such as rigidity,34

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osmotic fragility and deformability,35 which may justify the observed neutral effects of

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PS on the eryptotic process.

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Evaluation of hemolysis

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The hemolytic effects derived from our bioactive compounds are shown in Figure 3 B.

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No significant changes in the percentage of hemolytic cells were observed after

206

incubation with PS, though as expected and correlated with the increase in cell volume

207

(Figure 1 A), β-Cx showed a marked hemolytic effect, increasing the percentage of

208

hemolytic cells 52% versus the control. In the same way as previously seen in relation

209

to eryptosis, PS prevented hemolysis induced by β-Cx (55%). In line with our results

210

referred to PS, no statistically significant increase in hemoglobin concentration was

211

recorded after treatment during 48 h with the antioxidant apigenin (1-15 µM)36 or

212

whitaferin A at low concentration (1-2.5 µM).19 In addition, the absence of hemolytic

213

effects was also revealed from the individual sterol oxidation products (7α/β-hydroxy-,

214

α/β-epoxy- or 7-ketocholesterol) at physiological concentrations (1-7 µM, respectively);

215

however, cholestanetriol (2 µM) or a mixture of oxysterols at a concentration similar to


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that of our own study (20 µM) resulted in an 8% of lysed cells - though the different

217

hemolytic effects of the compounds assessed was not explained by the authors.20

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To our knowledge, only Briglia et al.22 have observed a significant increase in the

219

percentage of hemolytic erythrocytes (≈ 3-8%) after 48 h of treatment with fucoxanthin

220

(25-75 µM) (a compound structurally similar to β-Cx). According to the authors, β-Cx

221

may have a positive effect since it could allow the clearance of erythrocytes when these

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are infected with the malaria pathogen Plasmodium.

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Effect on cellular redox state: ROS and GSH

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Eryptosis is also stimulated by oxidative stress. Accordingly, in order to evaluate the

225

impact of PS and/or β-Cx upon the cellular redox state, intracellular ROS and GSH

226

contents were evaluated. As seen in Figure 4 A, none of the treatments with the

227

bioactive compounds promoted significant ROS overproduction, possibly due to their

228

potential antioxidant character.12 In the same way, PS did not produce significant

229

changes in GSH content (see Figure 4 B), which is consistent with the other previously

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evaluated parameters (annexin V, Ca2+ and hemolysis) and/or its neutral effects upon

231

the healthy erythrocyte membrane.34-35 β-Cryptoxanthin showed a significant decrease

232

in intracellular GSH content (10% versus the control), indicative of detrimental effects

233

upon redox state. This GSH depletion was restored when both bioactive compounds

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were co-incubated, showing a cellular response against oxidative stress. In agreement

235

with the behavior referred to the cellular redox state observed in our study, different

236

authors28,37 have reported the absence of increased ROS production with concomitant

237

GSH depletion, and suggest that erythrocyte ROS generation is a late event occurring

238

after severe GSH depletion.

239

Effect of PS and/or β-Cx on oxidative stress-induced eryptosis



240

Forward scatter, annexin V binding and cytosolic Ca2+

241

It has been reported that oxidant agents such as tBOOH are effective in inducing

242

eryptosis.27 We therefore evaluated the action of tBOOH at two concentrations (75 and

243

300 µM). As illustrated in Figure 5 (A and B), treatment with both tBOOH

244

concentrations produced an alteration in cell volume, particularly at 300 µM. The

245

analysis of the percentage of cells (Figure 5 C and D) revealed a limited effect at lower

246

induced stress, while an increase in the number of cells with FSC > 80 (associated to a

247

preliminary step of hemolysis) and FSC < 20 (cell size associated to eryptosis) at the

248

higher concentration (300 µM) was observed. The effect of this stressor upon

249

phosphatidylserine translocation showed that tBOOH at 75 and 300 µM led to a

250

significant increase in the percentage of annexin V-binding erythrocytes (1.3- and 2-fold

251

versus control, respectively) (Figura 6 A). Interestingly, dot plot graphics (FSC versus

252

annexin V) revealed that both erythrocyte populations (FSC < 20 or > 80) presented

253

phosphatidylserine translocation, being stronger under high stress levels (see Figure 6

254

B). Regarding intracellular Ca+2 analysis, an activation of Ca+2 influx in a dose-

255

dependent manner (26% and 78% versus control, respectively) was observed (see

256

Figure 7 A). In agreement with our results, other authors have reported that tBOOH at

257

300 µM is an excellent oxidative agent and inducer of eryptosis, promoting

258

phosphatidylserine translocation (15- to 55-fold, compared with untreated cells)27,38-39

259

and increasing the intracellular Ca2+ levels (36% compared with untreated cells).27

260

However, the exact mechanism is not well known yet. It has been reported that protein

261

kinase CK1 (casein kinase I) isoform α has a role in the regulation of erythrocyte

262

suicide- induced by tBOOH through modulation of cytosolic Ca2+ activity.40 Moreover,

263

other molecular pathways such as the heterotrimeric G protein subunit Gαi2,41 mitogen-

264

and stress-activated kinases MSK1 and MSK2,42 p38 mitogen-activated protein kinase


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(MAPK),43 which promote the eryptosis induced by hyperosmotic shock cannot be

266

ruled out.

267

Due to the oxidative stress induced by tBOOH, we evaluated the effect of our bioactive

268

compounds (PS and/or β-Cx). Pre-incubation during 48 h of erythrocytes with PS did

269

not modify eryptotic tBOOH action, though a non significant protective tendency at

270

high-stress concentration was observed (see Figure 6 A), in addition to a preventive

271

effect upon Ca+2 entry only under low-stress conditions. Regarding β-Cx, an increase in

272

cells with FSC < 20 (45% versus tBOOH 300 µM), with a concomitant decrease in cells

273

with FSC > 80 (8% versus tBOOH 300 µM), was observed (Figure 5 C and D). At high-

274

stress concentration, β-Cx produced a marked pro-eryptotic effect (annexin V assay) (3-

275

fold versus tBOOH 300 µM), without altering the intracellular Ca+2 levels. Moreover,

276

Figure 6 B shows that the presence of β-Cx increases erythrocytes population FSC > 80

277

(versus control) positive to annexin V, suggesting its role as a hemolytic and eryptotic

278

compound. Finally, co-incubation with both bioactive compounds (PS and β-Cx)

279

partially prevented the action of tBOOH and tBOOH plus β-Cx upon low-FSC cells

280

(Figure 5 C). Similarly to the situation observed without oxidative stress, co-incubation

281

with both bioactive compounds significantly (p < 0.05) blunted the increase in annexin

282

V-binding erythrocytes (38% versus tBOOH + β-Cx) (see Figure 6 A and B), and

283

tended to reduce Ca+2 influx promoted by β-Cx under high-stress conditions (tBOOH

284

300 µM) (see Figure 7 A).

285

Evaluation of hemolysis

286

As is shown in Figure 7 B, tBOOH treatment produced a limited hemolytic effect that

287

proved statistically significant (p < 0.05) only at 300 µM (18% higher versus control).

288

Therefore, considering the annexin binding results, it is concluded that tBOOH at low

289

concentrations (75 µM) induces erythrocyte death mainly via eryptosis through 13 ACS Paragon Plus Environment


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phosphatidylserine exposure, while at high concentrations (300 µM), eryptosis and

291

hemolysis occurred simultaneously. The induction of hemolysis by tBOOH at 300 µM

292

would explain the lack of effect on FSC reduction (see Figure 5), because hemolysis is

293

associated with an increase in swelling cells.1 Such swelling is possibly due to a

294

decrease in Na+/K+ ATPase activity in the erythrocytes, together with an increase in

295

Na+, loss of K+, depolarization and entry of Cl−, leading to an electrolyte increment that

296

promotes water entry accompanied by cell swelling.39 Pre-incubation with PS

297

completely prevented hemolysis produced by tBOOH. However, no preventive effect

298

on hemolysis was observed with β-Cx, producing a significant (p < 0.05) increase in

299

hemolytic erythrocytes under oxidative conditions of 24% and 59% versus tBOOH

300

control at 75 and 300 µM, respectively. Nevertheless, co-incubation with both bioactive

301

compounds yielded interesting results, since the presence of PS completely avoided

302

erythrocyte damage induced by tBOOH and tBOOH plus β-Cx, and even improved the

303

erythrocyte state, with hemolysis values lower than in the control (45% and 51%,

304

respectively).

305

Effect on cellular redox state: ROS and GSH

306

As illustrated in Figure 8 A, treatment with tBOOH 75 and 300 µM triggered significant

307

(p < 0.05) ROS overproduction, particularly at 300 µM (1.6- and 12-fold compared to

308

control, respectively), and only at lower induced stress (75 µM) was the addition of PS

309

and/or β-Cx to erythrocytes able to blunt ROS overproduction. Intracellular GSH

310

depletion (see Figure 8 B) was also lower at tBOOH 75 µM than at 300 µM, but a dose-

311

dependent relationship was not observed (15% and 22% decrease versus control at 75

312

and 300 µM, respectively). Pre-incubation with PS blunted GSH depletion produced by

313

tBOOH at 75 µM, but only a trend to recover GSH depletion was observed at 300 µM.

314

It is thus suggested that when erythrocytes are damaged by mild oxidative stress, PS are 14 ACS Paragon Plus Environment

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able to protect the cells against it. The fact that PS completely prevented GSH depletion

316

at lower stress concentrations, and partially prevented depletion at higher stress

317

concentrations, could suggest that the cell redox balance is preserved as a primary

318

event, since it has been observed that ROS generation is a late event in stressed

319

erythrocytes and that eryptosis can occur via primary GSH depletion independently of

320

ROS generation.28

321

On the other hand, the addition of β-Cx with tBOOH to the erythrocytes was not

322

effective in preventing GSH depletion with the lowest stress levels, and even marked

323

GSH depletion was observed at higher induced stress (300 µM). Therefore, the GSH

324

depletion induced by β-Cx could represent a key mechanism of its eryptotic effect,

325

mainly when stress was higher (Figure 8 B). Co-incubation with both bioactive

326

compounds (PS and β-Cx) showed no effect on GSH versus their respective tBOOH

327

control at 300 µM, except when co-incubated with tBOOH 75 µM - where the GSH

328

levels were completely restored, avoiding erythrocyte oxidative damage.

329

In conclusion, physiological serum PS concentration (22 µM) exerted no eryptotic or

330

hemolytic effect on human erythrocytes. However, under conditions of oxidative stress

331

(tBOOH) a cytoprotective effect upon the hemolytic process was observed, with partial

332

prevention of eryptotic cell death at low stress levels. In contrast, β-Cx (1 µM) produced

333

an increase in hemolytic and eryptotic cells - these effects being exacerbated in the

334

presence of induced stress. Co-incubation with both bioactive compounds showed the

335

presence of PS to mitigate eryptosis and hemolysis induced by β-Cx, with or without

336

induced oxidative stress. Considering the results globally, PS would help prevent the

337

pro-eryptotic and hemolytic effect induced by β-Cx and hydroperoxide–induced

338

oxidation – thereby possibly affording protection against diseases associated with

339

eryptosis, such as anemia, atherosclerosis, malignancy, obesity, diabetes, and chronic 15 ACS Paragon Plus Environment


340

inflammatory diseases, among disorders. It is important to highlight that this novel and

341

not previously described anti-eryptotic effect of PS (complementary to their well-known

342

cholesterol-lowering and anti-cancer effects) can be achieved in the range of

343

concentrations effective against tumor cells.14 This is of paramount relevance, since a

344

recent review44 has searched for substances which stimulate tumor cell apoptosis and by

345

the same token inhibit eryptosis - which may avoid side effects such as anemia found in

346

cancer patients subjected to chemotherapy. On the other hand, β-Cx exhibited pro-

347

eryptotic and hemolytic effects - a fact that could accelerate the death of erythrocytes

348

infected by plasmodium and help fight malaria. This study therefore opens a window

349

for new potential beneficial applications of two antioxidant phytochemicals (PS and β-

350

Cx) which are commonly present in well-balanced diets or in functional foods.

351

ABBREVIATIONS:

352

ANOVA: analysis of variance, CMFDA: 5-chloromethylfluorescein diacetate, β-Cx: β-

353

cryptoxanthin; DCFDA: 2´,7´-dichlorofluorescein diacetate; FSC: forward scatter,

354

GSH: glutathione; PS: phytosterols; ROS: reactive oxygen species; tBOOH: tert-

355

butylhydroperoxide.

356

FUNDING

357

This study belongs to national project AGL2015-68006-C2-1-R, financed by the

358

Ministerio de Economía y Competitividad (MINECO) and the Fondo Europeo de

359

Desarrollo Regional (FEDER), Spain. Andrea Álvarez-Sala Martín (ACIF/2015/251)

360

and Gabriel López-García (ACIF/2016/449) hold a grant from the Generalitat

361

Valenciana (Spain).

362

Notes: The authors declare no competing financial interest

363

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FIGURE CAPTIONS Figure 1. Effect of phytosterols (PS) and/or β-cryptoxanthin (β-Cx) at serum concentrations (22 and 1 µM, respectively) on the forward scatter (FSC) after 48h of incubation with human erythrocytes: (A) Geometric mean ± SD (n=3) of FSC; (B) Histogram of erythrocyte FSC in a representative treatment; (C) Arithmetic mean ± SD (n=3) of the percentage of erythrocytes with FSC < 20 (arbitrary units); (D) Arithmetic mean ± SD (n=3) of the percentage of erythrocytes with FSC ˃80 (arbitrary units). Different lowercase letters (a-d) indicate statistical significant differences (p < 0.05) among treatments. Figure 2. Evaluation of the (A) Phosphatidylserine exposure and (B) Dot plots of forward scatter (FSC) versus annexin V fluorescence exposure, after 48h incubation with phytosterols (PS) and/or β-cryptoxanthin (β-Cx) at serum concentrations (22 and 1 µM, respectively) on human erythrocytes. Data are expressed as mean ± SD (n=3). Different lowercase letters (a-c) indicate statistical significant differences (p < 0.05) among treatments. Figure 3. Evaluation of the (A) Ca2+ influx and (B) Hemolysis after 48h incubation with phytosterols (PS) and/or β-cryptoxanthin (β-Cx) at serum concentrations (22 and 1 µM, respectively) on human erythrocytes. Data are expressed as mean ± SD (n=3). Different lowercase letters (a-c) indicate statistical significant differences (p < 0.05) among treatments. Figure 4. Effect of phytosterol (PS) and/or β-cryptoxanthin (β-Cx) at serum concentrations (22 and 1 µM, respectively) on (A) Reactive oxygen species (ROS) production and (B) Intracellular reduced glutathione (GSH) content after 48h of incubation on human erythrocytes. Data are expressed as geometric mean ± SD (n=3).


Page 24 of 35

Page 25 of 35


Different lowercase letters (a-c) indicate statistical significant differences (p < 0.05) among treatments. Figure 5. Changes on forward scatter (FSC) under oxidative stress induced by tBOOH and potential preventive effect of pre-incubation for 48h with or without phytosterols (PS) and/or β-cryptoxanthin (β-Cx) at serum concentrations (22 and 1 µM, respectively) on human erythrocytes: (A and B) Histograms of erythrocyte FSC in a representative treatment with tBOOH at 75 and 300 µM, respectively; (C and D) Arithmetic mean ± SD (n=3) of the percentage of erythrocytes with FSC < 20 or with FSC ˃80 (arbitrary units), respectively. Different capital letters (A-C) indicate statistical significant differences (p < 0.05) among the different samples exposed to tBOOH at 75 µM and the lowercase letters (a-d) respect to tBOOH at 300 µM. Figure 6. Analysis of (A) Phosphatidylserine exposure and (B) Dot plots of forward scatter (FSC) versus annexin V fluorescence exposure, after 30 min of incubation with tBOOH (75 or 300 µM) with or without pre-incubation for 48h with phytosterols (PS) and/or β-cryptoxanthin (β-Cx) at serum concentrations (22 and 1 µM, respectively) on human erythrocytes. Data are expressed as mean ± SD (n=3). Different capital letters (A-B) indicate statistical significant differences (p < 0.05) among the different samples exposed to tBOOH at 75 µM and the lowercase letters (a-d) for tBOOH at 300 µM. Figure 7. Evaluation of (A) Ca2+ influx and (C) hemolysis after 30 min of incubation with tBOOH (75 or 300 µM) with or without pre-incubation for 48h with phytosterols (PS) and/or β-cryptoxanthin (β-Cx) at serum concentrations (22 and 1 µM, respectively) on human erythrocytes. Data are expressed as mean ± SD (n=3). Different capital letters (A-D) indicate statistical significant differences (p < 0.05) among the different samples exposed to tBOOH at 75 µM and the lowercase letters (a-d) for tBOOH at 300 µM.



Figura 8. Changes on (A) Reactive oxygen species (ROS) production or (B) Intracellular reduced glutathione (GSH) content after treatment with 30 min tBOOH at 75 or 300 µM with or without previous pre-incubation (48h) with plant sterols (PS) and/or β-criptoxanthin (β-Cx) at serum concentrations (22 and 1 µM, respectively) on human erythrocytes. Data are expressed as geometric mean ± SD (n=3). Different capital letters (A-C) indicate statistical significant differences (p80

1.00 % cells with FS C

or Î²-cryptoxanthin at physiological serum

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