Author Affiliations
Abstract
The study aimed to determine the effects of Cannabis sativa smoke on serum estrogen and progesterone levels in female Wistar rats. Forty (40) adult female Wistar rats weighing about 170-250g were used for this research work and divided into five (5) groups of eight (8) animals each based on average body weight. Control groups (A and B) were exposed to normal air and smoke from cigarette wrappers, respectively. Test groups (C, D, and E) were exposed to the smoke of completely burnt 1.0g C. sativa wrapped with 0.5g of sterilized wrapper once daily, twice daily, and thrice per day, respectively. All treated animals had one week and two weeks of exposure to the smoke of C. sativa. Twenty-four hours after the last administration for each week’s exposure, four animals in each group were sacrificed under chloroform anesthesia. The blood samples were collected through the femoral artery and analyzed for biomarkers. The study showed that the following administration of C. sativa extract at higher concentrations suppresses the generation of luteinizing hormone, lowering estrogen and progesterone levels. These impacts may alter the female menstrual cycle, posing a threat to female reproductive health.
Keywords
Cannabis sativa, Progesterone, Estrogen, Reproductive health, Wistar rats.
Introduction
Smoking is the most important public health problem. Many studies have proved its deleterious effects on many organ systems, mainly the respiratory, reticuloendothelial, and cardiovascular systems.[1] According to data from the World Health Organization (WHO), approximately 5 million people die globally each year from diseases caused by smoking, and if the trend continues, it is expected that by 2015, that number will be 10 million. Smoking results in more deaths each year than AIDS, alcohol, cocaine, heroin, homicides, suicides, motor vehicle crashes, and fires.[2]
Marijuana or cannabis, a substance made from the leaves and flowers of the C. sativa plant, is the most widely used illegal drug.[3] It is a popular drug of choice among young people, mainly due to its easy availability and low cost.[4] In the past two decades, cannabinoids in C. sativa (marijuana) have emerged as crucial mediators in a variety of pathophysiological conditions. In recent years, there has been a dramatic increase in the number of marijuana users and the long-term health consequences of marijuana use.[5]
C. sativa is a dioecious (having male and female flowers in different plants), green, leafy plant with characteristic opposite, typically seven-fingered, lance-shaped leaves; on dry, sandy, slightly alkaline soil, it can reach a height of over seven meters.[6] Glandular hairs develop, usually on the female flower, which secretes a resin. For commercial purposes, female plants are more significant than male plants since their fibers are thicker, they produce nourishing seeds[6], and they contain the psychoactive component tetrahydrocannabinol (THC).[7]
The cannabis plant contains more than 421 chemicals, 61 of which are cannabinoids.[8] Delta-9-tetrahydrocannabinol (Δ-9-THC) and Cannabidiol (CBD), the two main components of the C. sativa plant, have distinct symptomatic and behavioral effects.[9] Δ-9-THC is the primary psychoactive constituent of the C. sativa plant and is believed to be primarily responsible for its resulting cognitive effects, psychotic symptoms, and anxiety, as well as the addictive potential of smoked cannabis.[10] Worth noting is the fact that more than 2000 compounds are produced by pyrolysis during the smoking of cannabis[8], and they are represented by various classes of chemicals such as nitrogenous compounds, amino acids, hydrocarbons, sugar, terpenes, and simple fatty acids. These compounds altogether contribute to the unique pharmacological and toxicological properties of cannabis.[11]
Estrogen is the primary female sex hormone. The female reproductive system and secondary sex characteristics are developed and regulated by it.[12] Estrogens are metabolized in all vertebrates [12] as well as some insects.[13] Their presence in both vertebrates and insects suggests that estrogenic sex hormones have an ancient evolutionary history. Estrone, estradiol, and estriol are the three major naturally occurring forms of estrogen in females. Estetrol, a different kind of estrogen, is produced only during pregnancy.
Methodology
Study area: This study was carried out at Ambrose Alli University, Ekpoma, Esan-West Local Government Area of Edo State. The town is located at latitude 60.75IN and longitude 60.13IE with an estimated population size of 125,842 people.[14] The inhabitants are mainly students, civil servants, and farmers.
Extract preparation: The sample of C. sativa (marijuana) was obtained from the Nigeria Drug Law Enforcement Agencies (NDLEA), Benin Command, Benin, Edo State, Nigeria, and botanically identified and authenticated at the herbarium of the botany department, Ambrose Alli University, Ekpoma. was air-dried under standard laboratory conditions. A measure of 1.0 g of dried marijuana was wrapped with 0.5 g of sterilized wrapper following the protocol of[15] and burnt to produce the smoke extract.
Experimental animals: This experimental investigation was done following the standard humane animal care as outlined in the “Guide for the care and use of Animals in research and teaching”, as approved by the Institute of Laboratory Animal Resources, National Research Council, DHHS, Pub. No NIH 86 – 23.[16] The study was carried out using apparently healthy forty female Wistar rats weighing 170-250g. They were procured from the laboratory animal house, college of medicine, Ambrose Alli University Ekpoma, Edo State, and transferred to the experimental laboratory health affairs ventures, Ekpoma. All animals were acclimatized for two weeks before the treatment commencement. The rats were fed with standard rat chow at a recommended dose of 100 g/kg as advised by the International Centre for Diarrhoea Disease Research, Bangladesh (ICDDR, B), and drinking water was supplied ad libitum.
Experimental design and procedure: Forty adult Wistar rats were used for this study for a period of 4 weeks (two weeks of acclimatization, one week of exposure, and two weeks of exposure). The animals were allotted into five groups consisting of eight rats each, (n-8) and housed in five different clean cages of dimensions 33.0 × 20.5 × 19.0 cm placed in well-ventilated standard housing conditions (12 h light and 12 h darkness; temperature: 28 to 31°C; humidity: 50 to 55%)[17], and their cages were cleaned every day. All animals were observed for illness, abnormal behaviour, and physical anomalies. The study involved the use of both experimental and observational study designs. Groups (A and B) served as control groups while groups (C, D, and E) served as test groups.
Group A: Exposed to normal air for 5 minutes once per day
Group B: Exposed to the smoke of burnt 0.5g of sterilized wrapper for 5 minutes once per day
Group C: exposed to the smoke of burnt 1.0g marijuana wrapped with 0.5g of sterilized wrapper once per day (9.00 am) for 5 minutes each
Group D: exposed to the smoke of burnt 1.0g marijuana wrapped with 0.5g of sterilized wrapper twice per day (9.00 am, 1.00 pm) for 5 minutes each
Group E: exposed to the smoke of burnt 1.0g marijuana wrapped with 0.5g of sterilized wrapper thrice per day, 9.00 am, 1.00 pm, and 5.00 pm, for 5 minutes each
Termination of experiment: At the end of the experiment, the first set of four (4) animals from each group were sacrificed on day eight (1-week exposure) after administration of marijuana, while the second set of four (4) animals were collected on day fifteen (2 weeks exposure). Blood samples were collected through the femoral artery after anesthetizing the rat.
Sample analysis: The blood samples were allowed to clot and centrifuged at 1000 rpm for 15 minutes at room temperature to obtain serum. The serum supernatants were separated into sterile bottles and stored frozen. The serum active form of estrogen (estradiol) and progesterone levels of female Wistar rats were analyzed using an enzyme-linked immunosorbent assay (ELISA) as described.[18] based on the principle of competitive binding.
Statistical analysis: Statistical analysis was performed using one-way analysis of variance ANOVA. Experimental data were presented as ± Standard error of the mean (SEM). A P-value of <0.05 was considered statistically significant.
Results
Results showed no significant difference (p>0.05) in body weight among the groups at the baseline. For both 1 week and 2 weeks of exposure, there was a significant increase in the body weight of female Wistar rats treated with marijuana (groups C, D, and E) when compared with control rats (A and B), as shown in Table 1a.
| Phases | Group A Mean±SD Weight (g) |
Group A Mean±SD Weight (g) |
Group A Mean±SD Weight (g) |
Group A Mean±SD Weight (g) |
Group A Mean±SD Weight (g) |
F | P |
|
Baseline |
93.40±5.61 | 96.70±6.52 | 93.80±7.87 | 96.50±5.72 | 95.40±6.96 | 0.418 | 0.698 |
| Week 1 | 150.00±7.42 | 153.10±9.86 | 175.00±25.06 | 181.30±7.76 | 195.90±4.23 | 5.805 | 0.004 |
| Week 2 | 185.80±6.60 | 183.30±7.96 | 230.20±25.36 | 238.20±7.44 | 247.40±4.33 | 7.892 | 0.001 |
Table 1a: Body weight at baseline, week 1, and week 2 of Wistar rats fed with marijuana and control rats
Key: Mean±SD values with the same superscript are not statistically different from each other at p>0<0.0
For the weekly change in body weight, there was a significant increase in the body weight of groups (A, B, C, D, and E) Wistar rats with an increase at week 1 and week 2 when compared with baseline, as seen in Table 1b.
| Groups | Baseline weight(G) Mean±SD |
Week 1 weight(G) Mean±SD |
Week 2 weight(G) Mean±SD |
F | P |
| A (N=8) | 93.40±5.61 | 150.00±7.42 | 185.80±6.60 | 85.678 | 0.000 |
| B (N=8) | 96.70±6.52 | 153.10±9.86 | 183.30±7.96 | 88.169 | 0.000 |
| C (N=8) | 93.80±7.87 | 175.00±25.06 | 230.20±25.36 | 82.709 | 0.000 |
| D (N=8) | 96.50±5.72 | 181.30±7.76 | 238.20±7.44 |
92.521 |
0.000 |
| E (N=8) | 95.40±6.96 | 195.90±4.23 | 247.40±4.33 | 69.116 | 0.000 |
Table Ib: Weekly change in body weight of Wistar rats fed with marijuana and control rats
Key: Mean±SD values with the same superscript are not statistically different from each other at p>0<0.05
At 1 week and 2 weeks exposure, it was observed that there was a significant increase (p<0.05) in estrogen levels of Wistar rats in groups (C and D) when compared with the control groups (A and B) while there was a significant reduction in estrogen levels of rats in group E when compared with other groups (A, B, C, E) as seen in table 2a.
| Phases | Group A Mean±SD Estrogen (pg/ml) |
Group A Mean±SD Estrogen (pg/ml) |
Group A Mean±SD Estrogen (pg/ml) |
Group A Mean±SD Estrogen (pg/ml) |
Group A Mean±SD Estrogen (pg/ml) |
F | P |
| 1WK (N=4) | 32.36±5.28 | 32.64±5.07 | 103.96±6.05 | 148.00±8.43 | 19.60±3.66 | 95.631 | 0.000 |
| 2WKS(N=4) | 65.94±14.19 | 66.40±15.60 | 97.56±7.29 | 164.02±12.19 | 19.08±3.95 | 25.941 | 0.000 |
Table 2a: Serum level of estrogen in female rats treated with marijuana and control rats
Key: Mean±SD values with the same superscript are not statistically different from each other at p>0<0.05
For the weekly change in estrogen levels, there was a significant increase (p<0.05) observed in estrogen levels of groups (A, B, D, and E) Wistar rats at week 2 when compared with week 1. Groups (C and D) showed no significant difference (p>0.05) in the estrogen level at week 2 when compared with Week 1, as shown in Table 2b.
| Group | Week 1 Mean±SD Estrogen (pg/ml) |
Week 1 Mean±SD Estrogen (pg/ml) |
F | P |
| A (N=4) | 32.36±5.28 | 65.94±14.19 | 3.545 | 0.001 |
| B (N=4) | 32.64±5.07 | 66.40±15.60 | 2.171 | 0.032 |
| C (N=4) | 103.96±6.05 | 97.56±7.29 | 1.834 | 0.069 |
| D (N=4) | 148.00±8.43 | 164.02±12.19 | 2.075 | 0.040 |
| E (N=4) | 19.60±3.66 | 19.08±3.95 | 0.875 | 0.384 |
Table 2b: Weekly change in estrogen of Wistar rats fed with marijuana and control rats
Key: p>0<0.05 is significant
At week 1 and week 2 exposure, a significant increase (p<0.05) was observed in progesterone levels of Wistar rats in groups (C and D) when compared with control groups (A and B) while there was a significant reduction of progesterone level in group E when compared with other groups (A, B, C, and D) as seen in table 3a.
| Phases | Group A Mean±SD Estrogen (pg/ml) |
Group A Mean±SD Estrogen (pg/ml) |
Group A Mean±SD Estrogen (pg/ml) |
Group A Mean±SD Estrogen (pg/ml) |
Group A Mean±SD Estrogen (pg/ml) |
F | P |
| Week1(N=4) | 12.08±1.25 | 12.22±1.11 | 18.52±0.75 | 20.48±4.52 | 10.88±2.22 | 11.882 | 0.000 |
| Week2(N=4) | 12.26±1.74 | 11.96±1.53 | 21.44±2.56 | 18.18±4.01 | 9.78±2.24 | 89.256 | 0.000 |
Table 3a: Serum level of progesterone in female Wistar rats treated with marijuana and control rats
Key: Mean±SD values with the same superscript are not statistically different from each other at p>0<0.05
For the weekly change in progesterone levels of Wistar rats treated with marijuana, there was no significant difference (p>0.05) in groups (A, B, C, D, and E) between week 1 and week 2 exposure when compared as shown in Table 3b.
| Groups | Week 1 Mean±SD Estrogen (pg/ml) |
Week 1 Mean±SD Estrogen (pg/ml) |
F | P |
| A (N=4) | 12.08±1.25 | 12.26±1.74 | 0.040 | 0.968 |
| B (N=4) | 12.22±1.11 | 11.96±1.53 | 0.875 | 0.384 |
| C (N=4) | 18.52±0.75 | 21.44±2.56 | 0.942 | 0.074 |
| D (N=4) | 20.48±4.52 | 18.18±4.01 | 0.731 | 0.446 |
| E (N=4) | 10.88±2.22 | 9.78±2.24 | 0.247 | 0.802 |
Table 3b: Weekly change in progesterone of Wistar rats fed with marijuana and control rats
Key: p>0<0.05 is significant
Discussion
Two essential ovarian steroids that are essential to the female ovarian and menstrual cycles are estrogen and progesterone. Estrogen and progesterone work together to maintain the uterine lining for the implantation of fertilized eggs and the maintenance of the uterus’s function during pregnancy. This investigation investigated the effects of C. sativa smoking on the levels of estrogen and progesterone in female Wistar rats. In this study, there were variations in the levels of estrogen and progesterone among the rats treated with marijuana when compared with controls. A significant increase was observed in the level of estrogen and progesterone among the rats treated with 1.0g of marijuana once and twice per day for one week and two weeks, respectively, when compared with the control. This finding is in agreement with the work of[19], who reported an increase in the levels of these hormones in rats treated with marijuana extracts, but in sharp contrast with the work of[20], who reported a decrease in the levels of these hormones in marijuana-treated Wistar rats. These discrepancies may be due to the timing of marijuana administration relative to these rats’ ovulatory cycles.[21] Additionally, there was a significant reduction in the levels of estrogen and progesterone in group E (rats treated with the smoke extract of marijuana 3 times per day). This was in line with the findings of[18], who noted a time-dependent decrease in estrogen and progesterone hormone levels after marijuana use. Studies on female rodents have demonstrated that the chronic or acute administration of a high dosage of marijuana significantly decreases the levels of estrogen and progesterone by[22] inhibiting the synthesis of luteinizing hormone.
The findings from this work also showed an increase in the body weight of female Wistar rats treated with marijuana when compared with control rats. This was in agreement with the works of[23] Amaza, Maidugu, Zirahei, Numan, and Maris (2013) and[24], who in their various studies reported an increase in the body weight of rats fed with marijuana. The increase in body weight has been attributed to the fact that marijuana and its many chemical components (cannabinoids) as well as substances produced within the body that activate cannabinoid receptors (endocannabinoids) appear to exert unique influences on the regulation of feeding behavior.[25,26] state that endocannabinoids are important mediators and metabolic regulators in mammalian physiology, with diverse and ubiquitous modulating actions, including the regulation of body weight.[27] also reported that stimulation of the CB1 receptors in the mammalian cannabinoid system specifically increases food craving and enjoyment, and promotes the deposition of energy as fat into adipose tissues, which may be responsible for the increase in weight as observed in the present study.
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Acknowledgments
The authors recognize the contributions of the Nigerian drug law enforcement agencies (NDLEA) Benin Command, Benin, Edo State, Nigeria, for helping to secure C. sativa.
Funding
This project was self-funded.
Author Information
Corresponding Author:
Sadoh Odion Rosemary
Department of Molecular Laboratory
Centre for Diseases Control and Research, Federal Medical Centre, Asaba, Delta State, Nigeria
Email: odionsadoh@fmcasaba.org
Co-Authors:
Adesuwa Eidangbe
Department of Chemical Pathology
Ambrose Alli University, Ekpoma, Nigeria
Iyenoma Kelvin Oghogho
Department of Information Technology
Federal Medical Centre, Asaba, Delta State, Nigeria
Ilikannu Samuel Okwuchukwu
Department of Obstetrics and Gynecology
Federal Medical Centre, Asaba, Delta State
Authors Contributions
All authors contributed to the conceptualization, investigation, and data curation by acquiring and critically reviewing the selected articles. They were collectively involved in the writing – original draft preparation, and writing – review & editing to refine the manuscript. Additionally, all authors participated in the supervision of the work, ensuring accuracy and completeness. The final manuscript was approved by all named authors for submission to the journal.
Ethical Approval
This project was approved by the Ambrose Alli University ethical committee.
Conflict of Interest Statement
The authors have declared no conflicting interests.
DOI
Cite this Article
Sadoh OR, Adesuwa E, Iyenoma KO, Ilikannu SO. Assessment of Cannabis sativa (marijuana) Smoke on Serum Estrogen and Progesterone Levels of Female Wistar Rats. medtigo J Med. 2024;2(4):e30622426. doi:10.63096/medtigo30622426 Crossref
