Perillyl alcohol-induced hematological modulations: insights into human blood cells damage, eryptosis, and systemic perturbations

Abstract

Perillyl alcohol (POH) is a natural monoterpene with established antitumor activity. Although its anticancer efficacy is well-documented, its impact on normal blood cells, particularly red blood cells (RBCs), remains underexplored. Given the importance of RBC integrity in maintaining homeostasis, this study aims to investigate the hematological effects of POH, focusing on hemolysis, eryptosis, and systemic blood parameters. Erythrocytes collected from June-August 2023 were treated with POH (0.5–2.5 mM) for 24 hr at 37°C. Hemolysis was assessed using photometric assays. Eryptosis was detected by flow cytometry using annexin V, Fluo4/AM, and H₂DCFDA to quantify phosphatidylserine (PS) translocation, intracellular Ca²⁺, and oxidative stress, respectively. Complete blood count (CBC) parameters were also analyzed. POH induced dose-dependent hemolysis, elevated intracellular Ca²⁺, and significant PS externalization, indicating increased eryptosis. The hemolytic activity of POH was supported by marked increases in LDH, CK, and AST. Notably, polyethylene glycol (PEG) significantly attenuated hemolysis, suggesting a protective effect. In whole blood, POH reduced RBC count, hemoglobin, and hematocrit, while increasing RDW-CV. Reticulocyte profiling showed elevated immature reticulocyte fraction (IRF) and a medium fluorescence ratio (MFR). Moreover, POH induced leukopenia with immature granulocytes and platelet aggregation. POH disrupts RBC integrity, triggering hemolysis and eryptosis independently of oxidative stress. The observed hematological alterations underscore potential systemic toxicity and highlight the need for further preclinical evaluation to guide its therapeutic use in oncology.

INTRODUCTION

Perillyl alcohol (POH), also known as p-mentha-1,7-diene-6-ol or 4-isopropenyl-cyclohexenecarbinol, is a naturally occurring, non-nutritive dietary monoterpene (Chen et al., 2015). It is present in the essential oils of various plants, such as lavandin, mint, and cherries, and is biosynthesized through the mevalonate pathway (Loutrari et al., 2004). POH has been shown to selectively induce apoptosis in tumor cells without affecting normal cells (Belanger, 1998) through multiple mechanisms, including RAS signaling inhibition (Hohl and Lewis, 1995), interference with ion homeostasis (Garcia et al., 2010), apoptosis (Ariazi et al., 1999), angiogenesis inhibition (Loutrari et al., 2004), and oxidative stress (Ma et al., 2016). Its small size and amphiphilic nature facilitate diffusion across biological membranes, including the blood-brain barrier (Chen et al., 2021). Preclinical cell lines and animal models have demonstrated tumor regression in pancreatic (Stark et al., 1995), mammary (Haag and Gould, 1994), liver (Mills et al., 1995), lung (Lantry et al., 1997), colon (Reddy et al., 1997), and other models. Multiple phase I and II clinical trials have explored oral and nasal administration of POH in patients with advanced solid tumors (Durço et al., 2021). While oral POH administration showed limited clinical efficacy and was associated with dose-limiting gastrointestinal toxicity (Peczek et al., 2023), intranasal delivery, especially in the treatment of recurrent gliomas, has demonstrated promising results (da Fonseca et al., 2008; Santos et al., 2018). Despite growing evidence supporting its anticancer efficacy, it remains unknown whether POH modulates the lifespan of red blood cells (RBCs).

Eryptosis, the programmed cell death of erythrocytes, is characterized by membrane blebbing, cell shrinkage, increased intracellular Ca²⁺, and externalization of phosphatidylserine (PS) (Repsold and Joubert, 2018). Unlike hemolysis, which causes abrupt cell rupture, eryptosis is a controlled process that allows the clearance of damaged or dysfunctional RBCs by macrophages, preventing the release of toxic intracellular contents. Dysregulated eryptosis contributes to anemia and may reflect systemic toxicity (Tkachenko et al., 2025b).

Given the central role of RBC integrity in maintaining oxygen delivery and homeostasis, this study aims to evaluate its modulatory effects on RBCs, with a particular emphasis on its ability to induce hemolysis, eryptosis, and changes in hematological parameters. Additionally, this study explores the broader impact of POH on reticulocyte maturation, white blood cells, and platelet indices. The overarching goal is to provide critical insights into the safety and toxicity profile of POH, thus supporting its more effective therapeutic application.

MATERIALS AND METHODS

Experimental design

Solarbio Life Sciences (Beijing, China) provided all chemicals. A stock solution of POH at a concentration of 131 mM was prepared in dimethylsulfoxide (DMSO) and stored as aliquots at -80°C. Experimental cells were treated with POH at concentrations ranging from 0.5 to 2.5 mM for 6, 12, and 24 hr at 37°C. For the hemolytic and eryptotic assays, RBCs suspended in distilled water served as the positive control, whereas those suspended in standard Ringer buffer were used as the negative control. In addition to standard Ringer buffer (Jemaà et al., 2017), modified versions of this solution were employed, including those lacking Ca²⁺ or supplemented with 125 mM KCl, 150 mM urea, 283 mM sucrose, or 10% w/v polyethylene glycol 8,000 (PEG).

Blood collection

This study received ethical approval from the Ethics Committee of King Saud University (E-20-4544), and sample collection was conducted between June and August 2023. EDTA and heparin blood samples were collected from fifteen healthy volunteers who provided informed consent in compliance with the Declaration of Helsinki. The study subjects were 9 males and 6 females whose age ranged from 23 to 36 years). Erythrocytes were isolated by centrifugation at 3,000 RPM for 20 min and subsequently resuspended at 20% v/v in Ca²⁺-free Ringer buffer (Alfhili and Lee, 2021).

Hemolytic markers

The supernatants were harvested by centrifugation (13,000 RPM for 1 min) and hemoglobin levels in the supernatants were measured at 405 nm using the LMPR-A14 microplate reader (Labtron Equipment Ltd., Surrey, UK). The supernatants were also analyzed for levels of lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and creatine kinase (CK) using a BS-240Pro clinical chemistry analyzer (Mindray, Shenzhen, China). LDH measurement is based on the conversion of L-lactate to pyruvate and the reduction of NAD⁺ to NADH which reflects LDH activity. In the AST activity assay, the transamination of L-aspartate and α-oxoglutarate to oxaloacetate and L-glutamate by AST is accompanied by the concomitant oxidation of NADH to NAD⁺ which is proportional to AST activity. As for CK, the enzyme catalyzes the phosphorylation of ADP, in the presence of creatine phosphate, to form ATP and creatine. CK activity is then determined based on the rate of NADPH formation by hexokinase and glucose-6-phosphate dehydrogenase coupled reactions.

Cytosolic Ca²⁺

Intracellular Ca²⁺ levels were measured by a Northern Lights flow cytometer (Cytek, Fremont, CA, USA). A uniform suspension of control and treated cells (50 μL) were incubated in the dark at 37°C for 30 min with 5 μM Fluo4/AM prepared in a 5-mM CaCl₂-containing Ringer buffer (150 μL). Subsequently, 10,000 cells were analyzed for Fluo4 fluorescence intensity at 488/520 nm excitation/emission spectra, which reflects intracellular Ca²⁺ concentration (Rossi and Taylor, 2020).

PS translocation

Cells (50 μL) were incubated in the dark at room temperature for 10 min in a 5-mM CaCl₂-containing Ringer buffer with 1% annexin-V-FITC (150 μL). Following staining, 10,000 events were acquired and analyzed using flow cytometry at 488/520 nm excitation/emission spectra.

Oxidative stress

Uniform suspensions of control and treated (50 μL) RBCs were incubated with 5 μM 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) in 150 μL of a 5-mM CaCl₂-containing Ringer buffer for 30 min at 37°C away from light, then DCF fluorescence at 488/520 nm excitation/emission spectra was analyzed by flow cytometry (Alfhili et al., 2022).

Cellular morphology

Using flow cytometry, forward scatter (FSC) and side scatter (SSC) intensity values were simultaneously measured and expressed in arbitrary units (a.u.) to reflect size and granularity, respectively (Alfhili and Lee, 2021).

Ex vivo whole blood toxicity

Whole blood collected in EDTA was treated with 2.5% DMSO as a vehicle control or with POH at 2.5 mM for 24 hr at 37°C. Complete blood count (CBC), comprising RBC indices, reticulocyte indices, WBCs, and platelet indices, was evaluated using a Mindray BC-6200 hematology analyzer (Tkachenko et al., 2025b).

Statistical analysis

All measurements were recorded from three separate experiments utilizing matched cells for both control and treated groups. Statistical analysis was performed using Student’s t-test or one-way ANOVA with Prism 9 (GraphPad Software, Inc., La Jolla, CA, USA), with P-values less than 0.05 considered statistically significant.

RESULTS

POH induces concentration-dependent hemolysis

The hemolytic effect of POH was evaluated by exposing cells to concentrations ranging from 0.5 to 2.5 mM, and hemoglobin levels were measured in the supernatant. As shown in Fig. 1B, hemolysis increased significantly in a dose-dependent manner compared to the control, starting at 1 mM (P < 0.0001). Also, POH-induced hemolysis led to significant elevations in LDH (Fig. 1C), CK (Fig. 1D), and AST (Fig. 1E) activities.

Fig. 1

Effect of POH on hemolysis. (A) Molecular structure of POH. (B) Concentration- and time-dependent hemolytic activity of POH (0.5–2.5 mM for 6, 12, and 24 hr) in Ringer solution. POH-induced leakage of hemolytic markers: (C) LDH, (D) CK, and (E) AST. Data are presented as means + SEM (N = 3). ns indicates no statistical significance, while * (P < 0.05), ** (P < 0.01), *** (P < 0.001), and *** (P < 0.0001) as analyzed by one-way ANOVA. All endpoints were measured at 24 hr unless otherwise noted.

POH elevates calcium levels

A major trigger of RBC death is nucleation of Ca²⁺ (Tkachenko et al., 2025b). The influence of POH on intracellular Ca²⁺ levels was thus examined by labeling control and treated cells with Fluo4/AM following the previously described protocol. As indicated in Figs. 2A–2C, Fluo4 fluorescence and cells with increased Ca²⁺ significantly increased upon POH treatment (P < 0.05). We then evaluated the importance of Ca²⁺ to POH-induced hemolysis, by incubating the cells with POH in standard and Ca²⁺-free Ringer buffers, and found that Ca²⁺ deprivation significantly exacerbates POH toxicity (Fig. 2D, P < 0.05).

Fig. 2

Effect of POH on cellular Ca²⁺ content and eryptosis. (A) Representative histograms of Fluo4 fluorescence of control cells (gray) and treated (1.0–2.5 mM) cells (red). (B) Geomean of annexin-V-FITC fluorescence in control and treated (1.0–2.5 mM) cells. (C) Percentage of Fluo4-positive cells (1.0–2.5 mM). (D) Effect of Ca²⁺ availability on hemolysis in control and treated (2.5 mM) cells. (E) Representative histograms showing annexin-V-FITC fluorescence of control (grey) and treated (1.0–2.5 mM) cells (red). (F) Proportion of eryptotic cells expressed as a percentage following POH exposure (1.0–2.5 mM for 6, 12, and 24 hr). (G) Representative histograms illustrating DCF fluorescence in control and treated (2.5 mM) cells. (H) Percentage of cells that are DCF-positive (1.0–2.5 mM). (I) Effect of 1 mM of N-actyl-L-cysteine (NAC) on hemolysis in control and treated (2.5 mM) cells. Data are presented as means + SEM (N = 3). ns indicates no statistical significance, while * (P < 0.05), ** (P < 0.01), *** (P < 0.001), and **** (P < 0.0001) as analyzed by one-way ANOVA. All endpoints were measured at 24 hr unless otherwise noted.

POH promotes eryptosis

Beside hemolysis, another type of cell death is eryptosis which is characterized by PS translocation (Tkachenko et al., 2025b). As illustrated in Figs. 2E and 2F, the percentage of eryptotic cells significantly increased after treatment with POH at 1.5 mM (P < 0.05), 2 mM (P < 0.01), and 2.5 mM (P < 0.001) at 12 and 24 hr compared to the control. We were then prompted to probe the involvement of oxidative stress since it is an underlying mechanism of eryptosis (Tkachenko et al., 2025b). Interestingly, POH-induced eryptosis was independent of oxidative stress as detected by DCF fluorescence (Figs. 2G and 2H), which was confirmed by the lack of effectiveness of adding 1 mM of N-acetyl-L-cysteine (NAC) to the cells, as seen in Fig. 2I.

Effect of POH on cellular morphology

Eryptosis is accompanied by loss of volume due to membrane hyperpolarization and water loss (Tkachenko et al., 2025b). We estimated cell size by analyzing cells for FSC. The results in Figs. 3A–3C indicate significantly reduced FSC after treatment with 2 mM (P < 0.001) and 2.5 mM (P < 0.0001) POH. Similarly, exposure to 2 and 2.5 mM POH resulted in a significant increase in the proportion of shrunken cells compared to the control (P < 0.05), as evidenced in Fig. 3D. Next, to assess the requirement of membrane polarization to the toxic activity of POH, we incubated the cells with POH in 5 and 125 mM KCl to induce membrane depolarization. However, POH-induced hemolysis was not significantly altered in the presence of increasing concentrations of KCl (Fig. 3E), effectively ruling out volume loss as an essential mechanism.

Fig. 3

Effect of POH on RBC morphology. (A) Representative histograms of FSC of control cells (grey) and treated (2.5 mM) cells (red). (B) Original dot plots of FSC vs. annexin-V-FITC in control and treated (2.5 mM) cells. (C) Geomean of FSC in control and treated (1.0–2.5 mM) cells. (D) Percentage of cell shrinkage in control and treated (1.0–2.5 mM) cells. (E) Effect of KCl on hemolysis. Data are presented as means + SEM (N = 3). ns indicates no statistical significance, while * (P < 0.05), *** (P < 0.001), and **** (P < 0.0001) as analyzed by one-way ANOVA. All endpoints were measured at 24 hr.

PEG reduces POH-induced hemolysis

Many compounds have been reported to exert an anti-hemolytic effect (Tkachenko et al., 2025b), some of which were examined against POH. While urea (Fig. 4A) and sucrose (Fig. 4B) did not affect POH-induced hemolysis, PEG (Fig. 4C) offered significant protection (P < 0.05). Blocking casein kinase 1α (CK1α) activity with D4476 (Fig. 4D) was ineffective in reversing POH toxicity.

Fig. 4

Effect of modifiers on POH-induced hemolysis. Impact of (A) urea, (B) sucrose, (C) PEG, and (D) D4476 on POH-induced hemolysis (red and blue) compared to the control group (grey). Data are presented as means + SEM (N = 3). ns denotes no statistical significance, while * (P < 0.05) and **** (P < 0.0001) as analyzed by one-way ANOVA. POH was used at 2.5 mM and all endpoints were measured at 24 hr.

Effect of POH on RBC indices

An analysis of POH’s effect on RBCs in whole blood revealed multiple significant changes. POH (2.5 mM) significantly decreased RBC count, hemoglobin concentration, and hematocrit (P < 0.05), as shown in Fig. 5A. While a significant increase in red cell distribution width-coefficient of variation (RDW-CV) was noted (P < 0.05, Fig. 5A), there were no significant changes in mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), or mean corpuscular hemoglobin concentration (MCHC).

Fig. 5

Systemic toxicity of POH in whole blood and reticulocyte maturation. (A) RBC parameters, including RBC count, hemoglobin, hematocrit, MCV, MCH, MCHC, and RDW-CV. (B) Reticulocyte parameters include reticulocyte count, reticulocyte percentage, reticulocyte hemoglobin content, IRF percentage, LFR percentage, and MFR percentage. Data are presented as means + SEM (N = 3). ns denotes no statistical significance, while * (P < 0.05) and ** (P < 0.01) as analyzed by Student’s t-test. POH was used at 2.5 mM and all endpoints were measured at 24 hr.

Effect of POH on reticulocyte maturation

Our investigation of reticulocytes showed significant changes in their maturity indices. Although POH (2.5 mM) was associated with a significant decrease in overall reticulocyte count (P < 0.05), there was a significant increase in reticulocyte hemoglobin and immature reticulocyte fraction (IRF; P < 0.05), a decrease in low fluorescence reticulocytes (LFR; P < 0.05), and an increase in medium fluorescence reticulocytes (MFR; P < 0.05), as shown in Fig. 5B.

Effect of POH on WBCs

There was a significant reduction in WBCs (P < 0.0001; Fig. 6A) and neutrophils (P < 0.0001; Fig. 6A) with no effect on lymphocytes, monocytes, and eosinophils. POH was also toxic to basophils (P < 0.05; Fig. 6A). In contrast, there was a significant increase in immature granulocytes (P < 0.01; Fig. 6A).

Fig. 6

Effect of POH on white blood cell and platelet indices. (A) White blood cell parameters, including counts of leukocytes, neutrophils, lymphocytes, monocytes, eosinophils, basophils, and immature granulocytes. (B) Platelet indices, including platelet count, PCT, MPV, PDW-CV, P-LCC, P-LCR, and percentage of IPF. Data are presented as means + SEM (N = 3). ns denotes no statistical significance, while * (P < 0.05), ** (P < 0.01), and **** (P < 0.0001) as analyzed by Student’s t-test. POH was used at 2.5 mM and all endpoints were measured at 24 hr.

Effect of POH on platelet indices

A significant increase in the platelet count was noted upon POH treatment (P < 0.01; Fig. 6B), along with an elevated MPV (P < 0.05; Fig. 6B) and significant rises in platelet-large cell count (P-LCC) and ratio (P-LCR; P < 0.01; Fig. 6B). In contrast, plateletcrit (PCT), platelet distribution width-coefficient of variation (PDW-CV), and immature platelet fraction (IPF) remained unchanged.

DISCUSSION

The present study demonstrates that the antitumor concentrations of POH (Rajesh et al., 2003; Xu et al., 2004; Yuri et al., 2004) induce hemolysis in a concentration-dependent manner, suggesting its harmful impact on RBC membrane integrity and consequent release of intracellular components, including Hb, LDH, CK, and AST. Free Hb dimers, which act as strong oxidizing agents, are filtered through the glomeruli and subsequently accumulate within the renal tubules, where they contribute to both acute and chronic kidney injury. This damage is largely driven by the liberation of the heme moiety and free iron, especially under inflammatory conditions (Guerrero-Hue et al., 2018). In addition, circulating free Hb binds to and depletes nitric oxide reserves, resulting in impaired vascular relaxation and the development of ischemic tissue damage (Premont et al., 2020).

The disruption of the integrity of RBC membranes leads to uncontrolled influx of Ca²⁺, which is a key trigger for eryptosis (Lang and Qadri, 2012). Indeed, cytosolic Ca²⁺ activity was substantially increased in response to POH treatment. However, Ca²⁺ was not required for the hemolytic activity of POH, which indicates the possibility that other mechanisms have an impact. Similarly, KCl efflux does not appear to be essential for POH-induced toxicity, suggesting that other mechanisms may also contribute to its hemolytic effect. In addition, PEG has membrane-stabilizing and protective properties that can shield RBCs from the toxic effect of POH (Armstrong et al., 1997; Kameneva et al., 2003).

The percentage of eryptotic and shrunken cells significantly increased after treatment with POH, although without oxidative stress. The disruption of membrane asymmetry and the subsequent exposure of PS on the cell surface serve as key indicators of eryptosis, marking RBCs for clearance from the circulation (Tkachenko et al., 2025a). This process has been implicated in numerous pathological states, including metabolic syndrome, dyslipidemia, G6PD deficiency, hemoglobinopathies such as sickle-cell disease and thalassemia, hypertension, diabetes mellitus, and various forms of hemolytic anemia (Alghareeb et al., 2023; Lang et al., 2002; Lang et al., 2006; Zappulla, 2008). Importantly, the temporal relationship revealed by our time-course experiments (Figs. 1B and 2F) reveal that the two cell death modalities triggered by POH do not evolve in parallel. This divergence lends support to the notion that POH targets distinct, yet convergent, mechanisms that ultimately lead to membrane injury and loss of asymmetry. Collectively, eryptosis does not seem to be a precursor to hemolysis under the reported experimental conditions, and as noted in previous reports (Tkachenko et al., 2025a).

Erythrocytes are highly vulnerable to oxidative stress due to their constant exposure to oxygen, rich reservoir of polyunsaturated fatty acids, and the absence of mitochondria. As such, redox imbalance is a major trigger of erythrocyte cell death (Tkachenko et al., 2025a). However, our results demonstrate that POH does not increase intracellular ROS levels (Figs. 2G and 2H). Congruently, the contribution of oxidative injury does not seem to be essential for the full cytotoxic potential of POH as the addition of NAC did not rescue the cells (Fig. 2I). This redox-insensitive behavior points to additional mechanisms likely activated by POH, such as Ca²⁺ influx, K⁺ depletion, cytoskeletal disruption, and membrane pore formation.

The characterization of whole blood components revealed decreased RBCs, Hb, and hematocrit, along with increased RDW-CV, which can indicate anemia with anisocytosis. This is consistent with accelerating RBC loss and disruption of the normal population of RBCs, both in number and morphology, by eryptosis (Föller et al., 2008). Our Investigation of reticulocytes showed significant changes in their maturity indices. The elevated levels of IRF and MFR reflect suppressed reticulocyte growth.

The increase in platelet counts alongside the reduction in WBC count warrants further research and may reflect a systemic response to accelerated eryptosis or anemia. The elevated levels of immature granulocytes indicate a disruption in normal granulocyte maturation, implying that POH may impair key transcriptional pathways or delay responsiveness to growth signals necessary for effective granulopoiesis (Franchini et al., 2008; Khazal et al., 2023).

In conclusion, this study provides novel insights into the toxicity profile of POH, demonstrating that it can compromise RBC integrity, leading to hemolysis and eryptosis, characterized by the release of inflammatory intracellular components, PS exposure, and Ca²⁺ buildup without oxidative injury. The increase in platelets likely reflects clumping rather than an elevated cell count. Altogether, the current findings expand the current understanding of the toxicology of POH and provide a foundation for its continued exploration as a potential therapeutic candidate in cancer treatment. In particular, in vivo validation in animal models is essential to better contextualize the enclosed findings and guide further development of POH as a therapeutic agent.

ACKNOWLEDGMENT

The authors express their gratitude to the Ongoing Research Funding program - Research Chairs (ORF-RC-2025-3000), King Saud University, Riyadh, Saudi Arabia, for funding this research work.

Funding

This research was funded by the Ongoing Research Funding program - Research Chairs (ORF-RC-2025-3000), King Saud University, Riyadh, Saudi Arabia.

Conflict of interest

The authors declare that there is no conflict of interest.

Data availability

Data are available from the corresponding author on reasonable request, subject to King Saud University approval.

Author contributions

Conceptualization, M.A.A.; methodology, all authors; software, J.A.; validation, M.A.A. and A.M.B.; formal analysis, all authors; investigation, all authors; resources, M.A.A.; data curation, M.A.A. and A.M.B.; writing - original draft preparation, all authors; writing - review and editing, all authors; visualization, J.A.; supervision, M.A.A.; project administration, M.A.A.; funding acquisition, M.A.A and Y.A.A. All authors have read and agreed to the published version of the manuscript.

Ethical approval and consent to participate

The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of King Saud University (E-20-4544; 29 October 2022).

Patient consent for publication

Not applicable.

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