Enhancing Cytotoxicity of Tamoxifen Using Geranium Oil

Aromatherapy and plant-based essential oils are widely used as complementary and alternative therapies for various symptoms, including anxiety, mild mood disorders, and cancer-related pain. In a previous study, we developed an in vitro assay using immortalized hypothalamic neuronal cells (GT1-7 cells). In this study, we used this assay to investigate the effects of Geranium oil on the cytotoxicity of the oestrogen receptor (ER) antagonist: tamoxifen (TMX). The results showed that Geranium oil augmented TMX-induced cell death in a dose-dependent manner without directly reducing the viability of GT1-7 cells. Cotreatment with Geranium oil and ER agonist β-estradiol (E2) attenuated the inhibition of GT1-7 cell growth. Moreover, Geranium oil and geraniol, a major constituent of Geranium oil, showed weak agonist activity on ERα and ERβ with geraniol augmenting TMX-induced cell death similar to that observed in Geranium oil. Both compounds impair E2 activity. These data indicate that geraniol is an essential constituent of Geranium oil.


Introduction
Trace nutrients, such as vitamins and minerals, contained in plants are essential for human health. e essential oils used in aromatherapy are good for health. Aromatherapy is a folk remedy that encompasses the use of essential oils derived from various types of plant sources for a variety of applications and is widely used as a complementary or alternative therapy for symptoms including anxiety and depression [1].
Aromatherapy is performed by irritation to olfaction, or by application, inhalation, or oral administration of essential oils with the aroma component of plants and fruits. e chemical components in the essential oils bind with the receptors in the olfactory bulb causing these effects. ey stimulate the limbic system (the emotional center of the brain) which affect the physiological functions of the autonomic nervous, endocrine, and immune systems via hormones, neurotransmitters, and cytokines. Fragrant compounds and essential oils with sedative effects have influenced the motility of mice in inhalation studies [2]. Another report indicated that the odour of lavender ameliorated the influence of stress on brain mechanisms involved with the circadian rhythm of the autonomic nervous system activities and maintains their activity pattern during sleep [3]. Moreover, bergamot might be useful as an aromatherapeutic to minimise the symptoms of stress-induced anxiety, mild mood disorders, and cancer-related pain [4].
ere is a movement to complement the missing parts of Western medicine by using aromatherapy to improve medical methods (medical aromatherapy), with potential applications in obstetrics and gynaecological diseases, skin diseases, psychosomatic diseases, pain management, stress management, and upper respiratory tract infection treatment [1,2,[5][6][7]].
e olfactory system not only transmits odour information but also acts as an entry pathway for outside substances. Molecules can be transported from the olfactory epithelium to the olfactory bulb via retrograde axonal transport. Kanayama et al. [8] reported that metal ions administered intranasally are transported from the nasal cavity to the olfactory bulb via the olfactory nerve pathway and from there to other brain regions such as the hypothalamus and hippocampus. Another study found significant levels of fragrant compounds in the plasma after inhalation, indicating that the effects of aromatherapy result from direct pharmacological interaction rather than indirect central nervous system relays [2]. e components of essential oils applied to the skin using an aromatic bath or massage can be absorbed through the epidermis, dermis, hair follicles, and sebaceous glands. In cases of inhalation or oral administration, oils absorbed from mucous membranes in the nasal cavities and the gastrointestinal tract enter the blood and lymph vessels to potentially exert effects on the biological systems, such as the nervous system [1,2].
We previously showed that direct pharmacological interactions of several essential oils could be investigated using GT1-7 cell lines developed by genetically targeting the tumorigenesis of mouse hypothalamic neurons [9]. We found in a previous study that Geranium oil increased GT1-7 cell death induced by the oestrogen receptor (ER) antagonist tamoxifen (TMX) [9]. Geranium oil is commonly used as an element in aromatherapy owing to its many health benefits. It is used as a holistic treatment to improve physical, mental, and emotional health and has potential applications as an antidiabetic, anticancer, antibacterial, and antimicrobial agent [1]. Nakamura et al. reported that Geranium oil might be useful in ameliorating symptoms linked to premenstrual syndrome (PMS) [10]. β-Estradiol (E2) is involved in the regulation of gonadotropin-releasing hormone (GnRH) secretion in conjunction with ER. Geranium oil may affect GnRH secretion through the ER, contributing to the moderating effect of this oil on PMS. In this study, we investigated the components and mechanisms underlying the elevation of TMX-induced cell death in GT1-7 cells following the administration of Geranium oil. Essential oil is a mixture of various organic compounds and the effects of essential oils are likely due to the synergistic or competitive actions of these compounds, which need to be identified.

Materials and Methods
2.1. Reagents. Geranium oil was purchased from Tree of Life Co., Ltd. (Tokyo, Japan), TMX was purchased from Sigma-Aldrich (St. Louis, MO, USA), geranyl acetate was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), and other reagents were purchased from FUJIFILM Wako Pure Chemical Co., Ltd. (Osaka, Japan). ese compounds were diluted in dimethyl sulfoxide (DMSO) prior to use.

Cell
Culture. GT1-7 cells (provided by Dr. R. Weiner, University of California at San Francisco, CA, USA) were grown in Dulbecco's modified Eagle's medium/nutrient mixture F-12 Ham (DMEM/F-12) supplemented with 10% foetal bovine serum. After enzymatic digestion using trypsin, the cells were suspended in a serum-free medium and plated onto culture plates [11]. e cells were then cultured in a humidified incubator at 37°C and 5% CO 2 .

Cell-Viability Assay.
Cell viability was assessed as described previously [12]. Briefly, dissociated GT1-7 cells were plated onto 96-well culture plates at a concentration of 5 × 10 4 cells/well in 200 μL of culture medium. Following 24 h incubation, the cells were treated with Geranium oil at a final concentration of 5 μg/mL, 10 μg/mL, and 25 μg/mL, respectively. e major constituents of Geranium oil were used at the same concentration in the absence and presence of 5 × 10 −8 M E2. en, 0.5 μM TMX was immediately added to the medium. e final concentrations of the major constituents of Geranium oil are listed in Table 1. After 24 h of exposure, cell viability was quantified using the WST-1 assay with the Cell Counting Kit (Dojindo, Kumamoto, Japan). e absorbance of the treated samples was measured against a blank control using a microplate reader (iMark ™ microplate absorbance reader; Bio-Rad Laboratories, Hercules, CA, USA). e detection and reference wavelengths were 450 nm and 620 nm, respectively.

Analysis of the Components of Geranium Oil.
e major components of Geranium oil were analysed using a G1888 static headspace sampler connected to an Agilent 7890 gas chromatography (GC) system (Agilent Technologies, Mississauga, Ontario, Canada). GC analysis was performed according to the manufacturer's instructions, with minor modifications. e 20 mL headspace vials and aluminium crimp vial caps were purchased from Agilent Technologies. Geranium oil or its components (Table 1)  . e carrier gas (He) was administered at a flow rate of 1.33 mL/min, the oven temperature program ranged from 45-250°C at 7.79°C/min, and the FID and transfer-line temperatures were set to 250°C. e relative percentages of the compounds were calculated based on the peak areas from the FID data.

Nuclear Receptor Cofactor Assays.
e ERα/β agonist activity of Geranium oil or TMX was monitored using EnBio receptor cofactor assay system (RCAS) for ERα and ERβ kits (Fujikura Kasei Co., Ltd., Ibaraki, Japan) according to the manufacturer's instructions. We added 95 μL of ERα or ERβ solution to each coactivator (SRC1)-immobilised well. en, 5 μL Geranium oil (final concentrations: 10-25 μg/mL), geraniol (final concentrations are shown in Table 1), or (±) citronellol (final concentrations are shown in Table 1) was added in the absence and presence of E2 (final concentration: 1 nM) and TMX (final concentration: 25 μM). As a positive control, we added 5 μL of the 20 nM E2 and DMSO mixture to each appropriate well after adding the 95 μL ER solution. e plate was then incubated at room temperature for 1 h while shaking (∼800 rpm). All the wells were aspirated and washed with wash buffer included in the kit (three times). en, a 100 μM detection antibody solution was added to each well and incubated at room temperature for 30 min while shaking (∼800 rpm). All wells were again aspirated and washed (three times). en, the 100 μM TMB substrate was added to all the wells and incubated at room temperature for 20 min, after which a 100 μM stop solution was added to all the wells. e chromogen produced was measured at an absorbance of 450 nm using an iMark ™ microplate absorbance reader (Bio-Rad Laboratories).

Statistical Analyses.
All statistical evaluations were performed using a two-tailed student's t-test with the KareidaGraph v 4.0 Software (Synergy Software, PA, USA). A probability level (p) of <0.01 or <0.05 was considered significant.

Geranium Oil Enhances TMX Cytotoxicity.
e GT1-7 cell line is a useful model for investigating endocrine-disrupting chemicals, such as TMX [13]. In this, we investigated the effect of Geranium oil on TMX-induced cell death in GT1-7 cells (Figure 1). Treatment with 0.5 μM TMX significantly decreased the cell viability of the GT1-7 cells to 71.3 ± 16.5% (p < 0.01 vs. the control group). Administration of Geranium oil augmented TMX-induced cytotoxicity in a dose-dependent manner. Treatment with 5 μg/mL, 10 μg/ mL, and 25 μg/mL Geranium oil along with 0.5 μM TMX decreased the cell viability by 66.6 ± 12.1%, 56.6 ± 5.9% (p < 0.05 vs. the TMX 0.5 μM group), and 11.0 ± 9.3% (p < 0.01 vs. the TMX 0.5 μM group), respectively ( Figure 1). ere was no significant difference between the cell viabilities of the control and Geranium oil-treated groups ( Figure 1). ese results showed that Geranium oil augmented TMX-induced cytotoxicity without directly reducing the cell viability of GT1-7 cells.

Geranium Oil Displays Weak Agonist Activity on Both ERα and ERβ.
To investigate the involvement of the ER in the enhancement of TMX-induced cell death, we administered Geranium oil and/or TMX to GT1-7 cells in the presence and absence of 5 × 10 −8 M E2. e results showed that E2 increased the viability of the GT1-7 cells in a dose-dependent manner, with 5 × 10 −8 M E2 significantly increasing the cell viability by 162.6 ± 4.2% (maximum) (Figure 2). Moreover, E2 significantly inhibited TMX-induced cell death. Treatment with 5 × 10 −8 M E2 almost inhibited cell death induced by the 0.5 μM TMX (Figure 3). Interestingly, coadministration of Geranium oil with E2 significantly inhibited GT1-7 cell growth induced by E2 (Figure 3). e RCAS indicated that Geranium oil displayed weak agonist activity on both ERα and ERβ, with this activity increasing in a dose-dependent manner (Figures 4(a) and  4(b)). In contrast, TMX did not display ER agonist activity.
us, Geranium oil showed weak agonist activity on both ERα and ERβ.

Quantitative Analysis of Geranium Oil Components by
Gas Chromatograph. Various compounds are present in Geranium oil. In particular, citronellol, linalool, citronellyl formate, geraniol, and isomentone are present in high concentrations [14]. In this study, we quantified these components using GC. We detected peak derived from each compound according to their respective retention times (9.0 min for isomentone, 10.4 min for linalool, 11.4 min for citronellyl formate, 13.6 min for citronellol, and 14.6 min for geraniol); ethyl salicylate (14.0 min) was used as an internal standard. Calibration curves were generated for each compound to determine their respective concentration in the Geranium oil ( Figure 5 and Table 1).

Geraniol Enhancing Effect of Geranium Oil on TMX
Cytotoxicity. To clarify the mechanisms involved in the enhancement of TMX-induced cytotoxicity by Geranium oil, we investigated the effects of the respective constituents of Geranium oil identified using GC analysis on TMX-induced (0.5 μM) cell death. Using concentrations of each component equivalent to that contained in 25 μg/mL Geranium essential oil solution, we found that none of the components significantly altered the viability of GT1-7 cells in the absence of TMX relative to the control group (black and gray graph in Figure 6). When coadministered with 0.5 μM TMX, geraniol significantly reduced the cell viability from 67.6 ± 3.1% to 30.1 ± 2.3% as compared to TMX alone (white graph in Figure 6). None of the other components showed a significant effect on TMX-induced cytotoxicity. ese data suggest that geraniol is essential for the effect of Geranium oil on TMX-induced cytotoxicity in GT1-7 cells.
To confirm the involvement of ERs in the enhancement of TMX-induced cell death using geraniol, we investigated the interactions between ERα/β and geraniol (Figure 7). Geraniol was the only compound that bounded to both ERα and ERβ. Other constituents, such as citronellol, bounded with neither. Moreover, the ER agonist activity of geraniol was similar to that of Geranium oil. Despite their activities as ER agonists, coadministrating them with E2 indicated that both inhibited the ER agonist activity of E2. It is possible that   Figure 5: Chromatograms of major components of Geranium oil. e major components of Geranium oil were analysed using a G1888 static headspace sampler connected to an Agilent 7890 GC system. Geranium oil and its components are listed in Table 1, along with ethyl salicylate as an internal standard. Retention times and component concentrations were evaluated using commercial reagents as standards.  (table). After 24 h, the cell viability was analysed using WST-1 assay. Data represent the mean ± standard error of the mean (n � 6). e black graph represents the data of the untreated group.  Evidence-Based Complementary and Alternative Medicine geraniol competes with E2 as a partial ER agonist. ese data suggest that geraniol is an essential compound of Geranium oil that is involved in the enhancement of TMX-induced cytotoxicity.

Discussion
GT1-7 cells possess a number of neuronal characteristics, including neurite extension and secretion of GnRH. ey can also express receptors and neuron-specific proteins, including the E2 receptors (both ERα and ERβ subtypes), microtubule-associated protein 2, tau protein, neurofilament, synaptophysin, GABA A receptors, dopamine receptors, and L-type Ca 2+ channels [15]. ese properties make the GT1-7 cell line an excellent model system for investigating neurotoxicity and endocrine disruption [16,17]. Previously, we developed a convenient in vitro assay to evaluate the neuroendocrine effects of essential oils [9]. We revealed that in GT1-7 cells, Geranium oil enhances the cell death induced by TMX, using this assay. Moreover, we identified the active compound involved in this activity: geraniol. Neither Geranium oil ( Figure 1) nor geraniol ( Figure 6) significantly reduced the cell viability of these cells in the absence of TMX. In a previous study, we confirmed that Geranium oil did not potentiate the stimuli responsible for inducing cell death, such as oxidative stress or administration of high-doses of zinc or aluminium [9]. In this study, we verified that reduced cell viability was caused by secondary effects that enhanced TMX cytotoxicity and not by Geranium oil (geraniol) directly. TMX exerts antitumour effects via ERs. Previous studies have indicated that oestrogen is an important neurotrophic and neuroprotective factor [18]. In this study, TMX-induced cell death was alleviated by coadministrating the ER agonist E2 (Figure 3), suggesting that TMX might induce cell death by mediating the levels of ERs expressed in GT1-7 cells. Geranium oil contains geraniol. Howers et al. reported that geraniol was able to displace E2 from isolated ERα and ERβ; however, it did not show oestrogenic or antioestrogenic activity in an oestrogen-responsive human cell line [19]. is report showed that a possible partial interaction between geraniol and ER was identified using molecular graphics models [19], which suggested that geraniol affected ERmediated signal regulation by enhancing the effect of geraniol on TMX cytotoxicity. To test this hypothesis, we evaluated the ER activity of Geranium oil and geraniol using RCAS and found that both showed weak agonist activities on both ERα and ERβ. Coadministration of these materials with E2 inhibited the agonist activities of E2 on ERα and ERβ. ese results suggest that geraniol, which is an essential compound involved in enhancing the effect of Geranium oil on TMX cytotoxicity, competes with E2 as a weak ER agonist.
Oestrogen enhances and maintains reproductive function and the function of the central nervous system (CNS). It acts on the brain to elicit reproductive behaviour, including solicitation of the opposite sex and mating behaviour in female mammals [20]. It also regulates synaptic transmission, synaptic plasticity, and adult neurogenesis and behaviour [21][22][23]. Moreover, numerous studies have demonstrated that oestradiol, the most active oestrogen, exerts trophic actions on neurones and glial cells, promotes neuronal survival, and decreases neurodegenerative damage caused by a large variety of neuronal insults [24]. In our previous report, we proved that Geranium oil protects GT1-7 cells from stress related to neurological disorders, such as oxidative stress or administration of high dose of zinc [9]. In the CNS, the blood-brain barrier (BBB) restricts the translocation of molecules; however, the nasal cavity has attracted attention as a route to directly access the CNS  bypassing the BBB [25,26]. A previous study showed that nasal administration of wheat germ agglutinin to rats resulted in high concentrations of it in the olfactory bulb located at the tip of the brain via the olfactory nerve, which is connected to the nasal mucosa. Other studies reported that intranasal administration of insulin-like growth factor-1 and interferon β1b to rats and monkeys quickly distributed them to the olfactory bulb and brain stem via the trigeminal nerve and olfactory nerve [27][28][29]. Geranium oil contains geraniol, which is said to exhibit oestrogenic activity [19]. It is possible that nasal inhalation of Geranium oil results in geraniol transport directly into the CNS, resulting in its associated activity. e enhancing effect of Geranium oil on TMX cytotoxicity was also observed in another ER positive cell line such as human breast cancer cell line MCF-7 cells ( Figure S1). TMX blocks oestrogen-neuronal binding on tumour surfaces to reduce tumour growth and proliferation in patients with breast cancer. In light of mounting evidence in the role of oestrogen in the maintenance of specific dimensions of cognitive function, these patients have a greater lifetime risk for cognitive impairment because of reduced oestrogen levels because of hormone therapy with TMX [30]. Geranium oil (and its component geraniol) is a relatively safe and nontoxic substance, when combined with TMX can potentially enhance its efficacy or reduce the required dosage. Moreover, we found that Geranium oil and its component geraniol have a weak oestrogenic effect. Shinohara et al. reported that exposure to Geranium oil increased the concentration of salivary oestrogen [31]. ese findings suggest that Geranium may be useful for controlling the side effects of TMX caused by lower oestrogen levels. erefore, the complementary use of essential oils or their active compounds with TMX can reduce the risk of cognitive impairment related to hormone therapy.

Conclusion
We showed that Geranium oil enhances TMX-induced cytotoxicity in GT1-7 cells as a partial ER agonist, with the essential compound associated with this effect being geraniol. Oestrogen is involved in the maintenance of reproductive function, brain function, and prevention of dementia.
e nasal cavity has attracted attention as a noninvasive route for delivering substances directly to the CNS while bypassing the BBB. Nasally aspirated Geranium oil (and geraniol) might affect GnRH secretion through the ER, contributing to the treatment of neurological diseases, such as dementia. ese results suggest that Geranium oil can be used for clinical applications, including supplementation with hormone therapy to alleviate the side effects of TMX in the CNS.
Data Availability e data that support the findings of this study are available from the corresponding author, Dai Mizuno, upon reasonable request.

Conflicts of Interest
All authors declare that there are no conflicts of interest.