Association of Early Pubertal Onset in Female Rats With Inhalation of Lavender Oil

Background Central precocious puberty (CPP) is caused by early activation of the hypothalamic–pituitary–gonadal axis but its major cause remains unclear. Studies have indicated an association between chronic environmental exposure to endocrine-disrupting chemicals and pubertal onset. Essential oil is widely used in homes worldwide for relief of respiratory symptoms, stress, and/or sleep disturbance. Methods To evaluate this association, we compared the hormone levels and timing of vaginal opening (VO) in female rats exposed to lavender oil (LO) through different routes (study groups: control, LO nasal spray [LS], and indoor exposure to LO [LE]) during the prepubertal period. The body weights of the animals were also compared every 3 days until the day of VO, at which time gonadotropin levels and internal organ weights were assessed. Results The LS group showed early VO at 33.8 ± 1.8 days compared with the control (38.4 ± 2.9 days) and LE (36.6 ± 1.5 days) groups. Additionally, luteinizing hormone levels were significantly higher in the LE and LS groups than those in the control group. Body weights did not differ significantly among the groups. Conclusion Inhalation exposure to an exogenic simulant during the prepubertal period might trigger early pubertal onset in female rats. Further evaluation of exposure to other endocrine-disrupting chemicals capable of inducing CPP through the skin, orally, and/or nasally is warranted.


INTRODUCTION
Central precocious puberty (CPP) describes the early activation of the hypothalamicpituitary-gonadal (HPG) axis, which leads to the rapid progression of bone age, early menarche, reduction in final adult height, and the appearance of secondary sexual characteristics before 8 and 9 years of age in girls and boys, respectively. 1 Traditionally, CPP is accompanied by intracranial lesions, including optic glioma, pilocytic astrocytoma, hydrocephalus, Rathke's cleft cyst, and pituitary adenomas, in 40-90% of boys and < 10% of girls. 2,3 The gonadotropin-releasing hormone (GnRH) stimulation test is used for diagnosing CPP, and the basal luteinizing hormone (LH) level is considered as a valuable tool to assess pubertal state. 4 CPP treatment was introduced in 1980, and dosing of a recombinant GnRH agonist every 4 weeks or 3 months leads to increases in the final adult height and delayed menarche. 2,3 Improving the final adult height of children with CPP is one of the major issues during treatment. 5, 6 However, the incidence of precocious puberty is rapidly increasing, and examination and treatment of this condition are becoming a major burden because of the associated medical expenses, although the cause of this condition remains unknown. 2,3 Environmental hormones, i.e., endocrine-disrupting chemicals, were recently suggested to contribute to the onset of puberty in childhood, 7,8 and animal studies demonstrated that endocrine-disrupting chemicals accelerate pubertal onset. 9,10 Additionally, previous reports showed that lavender oil (LO) and tea tree oil are associated with prepubertal gynecomastia in boys. 11,12 Moreover, cases of premature thelarche that resolved after cessation of exposure to lavender-containing fragrance have been reported, 13,14 and an in vitro study showed that components of LO, including linalool and linalyl acetate, activate estrogen-related gene expression in human cell lines. 13 However, studies of the absorbance of these materials in sufficient amounts and their effect on breast growth have not been performed. A previous study suggested that smell of sense can be transmitted to the central nervous system, thereby facilitating the bypass of inhaled molecules via the nasal pathway of the blood-brain barrier. 15 There are several opportunities for inhalation of numerous endocrine-disrupting chemicals from estrogenic sources in cosmetics, perfumes, air fresheners, and scented candles/diffusers using essential oils, which can directly affect olfactory stimulation of the neuroendocrine system. Since some studies suggested that essential oils may have efficacy against the coronavirus disease 2019 and its inflammatory complications, 16-18 the interests for home therapy using essential oils are more increasing.
In this study, we tested whether continuous inhalation of LO affects early gonadotropin activation and precocious puberty. However, it is difficult to limit or measure the number of EDCs to nasal exposure in human. Thus, we investigated the effects of continuous inhalation of LO on pubertal onset and gonadotropin hormone levels in an animal model and compared them to control conditions.

Animals and experimental design
To obtain study animals, rats were bred using male and female Sprague-Dawley rats. From birth onward, we maintained an indoor temperature of 22°C (humidity: 30-70%) and controlled illumination (12-hour light/dark cycle) to allow breeding in a constant environment along with free access to water and food. On day 18 after birth, we identified 15 immature females and randomly divided them into three groups: olfactory stimulation groups 1 and 2 and a control group (n = 5/group). We used 100% pure LO obtained from Lavandula angustifolia (NOW Foods, Bloomingdale, IL, USA) for all experiments. Group 1 was treated by indoor exposure to LO (LE) via an LO diffuser in the cage using an LO-soaked puff (changed daily) along with daily exposure to 0.9% NaCl spray. For Group 2, LO was administered as a nasal spray of aromatic LO (LS) once daily. The control group was treated with a single exposure to a nasal spray (0.9% NaCl) daily. The dose of one spray of LO or 0.9% NaCl ranged from 72-125 µL. The body weight of the animals was measured every 3 days from postnatal day (PND) 18 to the day of vaginal opening (VO).

Analysis of VO
All study groups were evaluated for VO time as an indicator of pubertal initiation at a fixed time (09:00 hour) daily. The day of VO was recorded, and VO timing was compared between the three study groups.

Euthanasia and hormone assays
After VO was observed in each rat, we measured serum LH, follicle-stimulating hormone (FSH), and estradiol levels to compare hormone concentrations between study groups. The endpoint of the experiment was defined as VO occurring in the last rat. For this process, truncal blood was collected into ice-cold ethylenediamine tetraacetic acid-containing tubes after decapitation, after which the tubes were centrifuged, and plasma samples were collected and stored at −20°C until analysis. The plasma levels of LH, FSH, and estradiol of each rat were measured using enzyme-linked immunosorbent assay kits (cat. No. MBS764675, MBS2502190, and MBS263850, respectively; MyBioSource, Inc., San Diego, CA, USA) according to manufacturer's instructions.

Measurement of organ weight
After euthanasia, we measured the weight of the ovaries, spleen, kidneys, and liver. The organ weight was then modified by body weight and presented as tissue weight per 150 g body weight.

Statistical analysis
Data were presented as the mean ± standard deviation, and statistical analyses were performed using SPSS software (v.26.0; SPSS, Inc., Chicago, IL, USA). Statistical significance was determined by Kruskal-Wallis test and one-way analysis of variance for multiple-group comparisons and Mann-Whitney U test for comparisons between two groups. Statistical significance was defined at P < 0.05.

Ethics statement
The procedures used and the care of animals were approved by the Institutional Animal Care and Use Committee in Southwest Medi-Chem Institute (approval No. SEMI-20-001).

Effect of olfactory exposure to LO on VO and pubertal onset
VO occurred earlier in the LE (33.8 ± 1.8 days) and LS (36.6 ± 1.5 days) groups than in the control group (38.4 ± 2.9 days) ( Table 1), and VO in the LE group occurred significantly earlier than in the control group (P = 0.014) and LS group (P = 0.032), respectively. However, there was no significant difference between the LS and control groups with respect to VO timing (P = 0.151). Almost all rats in the LE group experienced VO at 33 days, and the control group mostly showed VO between 38 and 41 days (Fig. 1).

Measurement of body and organ weights
Measurement of the body weight of rats in the control, LE, and LS groups every 3 days from PND 18 until VO revealed no significant differences among three groups ( Fig. 2 and Table 3). The weights of the ovaries, liver, and spleen after VO showed no significant differences among three groups; however, the weight of the kidneys per 150 g body weight increased significantly after VO in the LE group (1.926 ± 0.154 g) compared with that in the control (1.664 ± 0.077 g; P = 0.009) and LS (1.694 ± 0.154 g; P = 0.017) groups ( Table 4).

DISCUSSION
In this study, we found that persistent exposure to LO is associated with the HPG axis activation and early pubertal onset. We observed early VO in rats persistently exposed to LO as compared with that in the control group.  115.4 ± 7.6 a g 110.2 ± 4.9 g 110.8 ± 5.7 g Data are presented as the mean ± SD (n = 5). PND = postnatal day, LS = exposure to lavender oil as a nasal spray, LE = exposure to diffused lavender oil. a Not significant. Additionally, serum LH and FSH levels were significantly higher in the LE and LS groups than in the control group. The LH level has been considered as a golden marker of pubertal status, whereas the estradiol level showed a fluctuation during the day and could be low even in the pubertal period. 22-25 Chronic and persistent exposure to estrogens could also affect gonadotropin activation. Chronic exposure to sex hormone in cases of peripheral precocious puberty, including congenital adrenal hyperplasia or McCune-Albright syndrome, could lead to the secondary CPP, and these patients require to be treated with the GnRH agonist. 26,27 Previous studies showed that estrogenic effect of LO affected the premature thelarche in girls and gynecomastia in boys. 11-14 To the best of our knowledge, no previous studies have reported an association between pubertal onset and persistent olfactory exposure to LO in an animal model. Nasal inhalation is an important source of iatrogenic sex-hormone exposure, and olfactory exposure to LO may result in LO delivery to the central nervous system and bloodstream to induce an iatrogenic effect of estrogen.
Several studies have shown that LO is effective at reducing menopausal symptoms and supporting healthy sleep. 28, 29 Additionally, previous studies reported that LO does not increase estrogen levels in adults 30 ; in contrast, in the present study, we could not conclude that LO exposure did not affect estrogen activity. Although we observed no differences in estradiol levels between the LE and control groups, the LE group showed early pubertal onset and significantly increased LH and FSH levels. Furthermore, the LS group showed no significant differences in VO compared with the control group. Therefore, the amount and persistence of LO exposure may determine pubertal onset.
A previous animal study reported that percutaneous injection of LO when performing uterotrophic assays on immature rats results in significantly reduced body weight gain after 3 days compared with that in the control group and a group administered 17α-ethinyl estradiol. 31 However, the authors only assessed weight gain and organ-weight-to-terminalbody-weight to evaluate the presence of an estrogenic effect, and did not compare hormone levels or VO timing. The different administration modalities may have been responsible for the reported differences in body weight gain between the LO injection group and the group undergoing oral estrogen administration. In the present study, we found no differences in organ weight, including the ovaries, and body weight gain between groups, despite the apparently different VO and gonadotropin levels. Interestingly, the kidney tissue weight was significantly increased only in the LE group, supporting an association between physiological LO-specific effects and endocrine effects. A recent study showed that LO exposure affects renal restoration in a dose-dependent manner by decreasing antioxidant signals and inflammatory cytokine levels, as well as by inhibiting apoptosis. 32 In the present study, we measured neither renal function nor nephron numbers and used only renal tissue weight as an indicator of the positive effect of LO exposure. Therefore, increased renal tissue weight may indicate a renal burden related to LO excretion.
Four studies have reported a total of 11 pediatric patients (seven males and four females) showing premature thelarche in females (age range: 14 months to 7 years and 9 months) and 6/9 https://jkms.org https://doi.org/10.3346/jkms.2022.37.e9 The Effect of Lavender Oil on Pubertal Onset gynecomastia in males (age range: 4 years and 5 months to 10 years and 1 month) after using an LO-containing product. 11- 14 Ramsey et al. 13 reported that patients showed symptomatic improvement after discontinuation of LO exposure accompanied by no abnormal laboratory findings. These reports suggest the estrogenic effect of topical preparation of LO. Additionally, in vitro studies showed that LO (or the LO components linalool and linalyl acetate) exerts an estrogenic effect by stimulating α-transcription of the estrogen receptor. 11, 13 The peripheral hormones or signals transmitting to GnRH neurons may lead to GnRH secretion and stimulation of pituitary gonadotropins and gonadal sex steroids. 8 Given our observation of early activation of the HPG axis after olfactory exposure to LO, further investigation of the effect of LO on gene activation related to GnRH synthesis or secretion may explain the associations between early activation of the HPG axis and LO exposure.
We focused on the effects of LO exposure through olfactory stimulation and not via oral ingestion or topical application. One limitation of this study is its small sample size; thus, further studies are required to validate the findings. We observed significant elevation of gonadotropin levels not only in the LE group but also in the LS group, suggesting that repetitive olfactory exposure affected the manifestation of LO-specific physiological effects. Several studies reported that essential LO affects anxiety when administered via the oral or nasal routes. 33-35 However, LO exposure via oral ingestion in food is not as common as skin absorption of various LO-containing cosmetic products or LO inhalation. Nevertheless, inhalation of environmental LO is difficult to quantify in humans, and epidemiological surveillance data for the sole effect of LO inhalation in children undergoing precocious puberty are unavailable. Furthermore, the frequency and duration of exposure to LO inhalation are highly variable, and the effect of LO following skin exposure on children remains inconclusive because of the variable amounts of LO to which the skin is exposed, and difficulties associated with follow-up to assess long-term effects. 36 This study showed the effect of olfactory stimulation by LO on the early onset of puberty. These results suggest that avoidance of LO exposure to minimize unnecessary iatrogenic estrogen effects from fragrances, diffusers, and perfumes can prevent early stimulation of the HPG axis, particularly in younger children. Further in vitro evaluation of LO-related effects on central kisspeptin signaling may reveal the mechanisms associated with early activation of the HPG axis by persistent olfactory exposure to LO.