SSR Inst. Int. J. Life Sci., 6(4):
2594-2600,
July 2020
Microanatomical
and Hormonal Studies of the Effects of Aqueous Cannabis sativa Leaf Extract on the Testis of Adult Wistar Rats
Ignatius Ikemefuna Ozor1,
Onyinye Mary Ozioko2, Uche Sebastine
Ozioko3, Christian Ejuiwa Mba2*, Ifeanacho Ezeteonu
Abireh3, Offor Chukwuebuka Blessed4
1Senior Lecturer, Department of Anatomy, College of Medicine, Enugu
State University of Science and
Technology, Enugu, Nigeria
2Assistant
Lecturer, Department of Anatomy, College of Medicine, Enugu State University
of Science and Technology, Enugu,
Nigeria
3Lecturer II,
Department of Anatomy, College of Medicine, Enugu State University of Science and Technology, Enugu, Nigeria
4Researcher,
Department of Anatomy, College of Medicine, Enugu State University of Science and Technology, Enugu, Nigeria
*Address for Correspondence: Mr. Christian Ejuiwa Mba, Department of
Anatomy, College of Medicine, Enugu State University of Science and Technology, Enugu, Nigeria
E-mail: dogo4all@yahoo.com
ABSTRACT- Background:
Cannabis sativa is a psychoactive drug
from the green, leafy Cannabis plant used
in Asian traditional medicine for the treatment of varieties of diseases
including inflammation, nausea, headache, hematochesia, diarrhea and alopecia
due to its cannabinoid and tetrahydrocannabinol properties.
Methods:
Thirty-six adult (36) adult male wistar rats divided into four groups (Groups
A, B, C & D) were used for this study. Group A was given normal saline, B
200 mg/kg, C 500 mg/kg & D 800 mg/kg of C.
sativa orally for 14 days. Ichroma
quantitative testosterone method (fluorescence
immunoassay) was used to determine testosterone in serum by screening and
monitoring androgen levels
Results:
Serum testosterone, luteinizing hormone (LH) and
follicle-stimulating hormone (FSH) levels decreased significantly (p<0.05)
in the experimental groups compared to the control especially for group D (800
mg/kg). Testicular parenchyma showed degeneration of sertoli cells as
well as a decrease in spermatogenic cells and sperm motility.
Conclusion:
Cannabinoid had negative effects on sertoli cells; sperm function and decreases
luteinizing hormone, follicle-stimulating hormone and testosterone levels in adult
wistar rats.
Key
Words: C. Sativa, Cannabinoid, Fertility,
Hormone, Sertoli cells
INTRODUCTION- Cannabis, also known as marijuana among other names [1] is a psychoactive drug from the Cannabis plant used primarily for medical or recreational purposes [2]. The main psychoactive component of cannabis is tetra hydro cannabinol (THC), which is one of the 483 known compounds in the plant [3] including at least 65 other cannabinoids. Cannabis can be used by humans by smoking, vaporizing, ingested as food, or as an extract [4].
Cannabis has mental and
physical effects on individuals who ingest it ranging from "high", or stoned feeling, general
change in thought and perception, difficulty concentrating, impaired short-term
memory, altered sense of time, impaired body movement, relaxation [5] and a sharp increase in appetite, otherwise known as
"munchies". The onset of effects is felt within few minutes when
smoked, and about 30 to 60 minutes when cooked and eaten [6]. The effects usually last
for about two to six hours post-ingestion, depending on the quantity or dose
consumed [6].
At high doses, mental
effects sometimes include psychosis, delusions, hallucinations, paranoia, and
ideas of reference, sometimes with anxiety and panic [7]. Its
physical effects include increased heart rate, difficulty breathing, nausea,
and behavioral problems in children whose mothers used cannabis during
pregnancy. Short-term side effects may include a dry mouth, red eyes, and
feelings of paranoia or anxiety [8]. Long-term
adverse effects may include addiction, decreased mental ability in those who
started regular use as adolescents, chronic coughing, and susceptibility to
respiratory tract infections [9]. Heavy,
long-term exposure to C. sativa may
have biologically-based physical, mental, behavioral and social health
consequences and maybe "associated with diseases of the liver, lungs,
heart, and vasculature" [10].
Drug abuse has been shown to affect gonadal hormones in an
unusual physiological phenomenon [11] and quite several studies have examined the effects of cannabis smoking
on the reproductive system. Cannabis has been reported to cause downregulation
of hypothalamic-pituitary-gonadal axis, endocrine disruption, and
hyperprolactinemia [12]. Significant shrinkage of tubular diameter
and detrimental changes in the seminiferous epithelium of testis with resulting
lowered serum testosterone and pituitary gonadotropins (FSH and LH) levels at
low doses [13]. Grossman [14] reported
that Cannabis affects ‘swimming behaviour’ of sperm but
the fertility effects associated with Cannabis is unclear. Therefore this work
is aimed at investigating the histochemical effects of aqueous C. sativa leaf extract on the testis of
adult wistar rats.
MATERIAL
AND METHODS
Amscope 5.0 digital microscope was used to make the micrographs after processing the testis in the histology laboratory.
Plant Materials- The leaves of the plant, C. sativa were harvested from plants grown in Kwale, Delta State, Nigeria with approval of the National Drug Law Enforcement Agency (NDLEA). The leaves were identified by a crop scientist (Dr. Ejike Ikenganyia) of the Department of Agronomy and Ecological Management, Faculty of Agriculture and Natural resources Management, Enugu State University of Science and Technology, Agbani, Enugu, Nigeria on October 2019.
Preparation
of Cannabis extract- The sample was dried in air for 7 days and after complete
drying was crushed in the form of fine powder with a super Sony Japan electric blender. 200 g of dried plant extract was immersed in 2000 mls of water and was evaporated
with a Soxhlet extractor to yield 21.2 g of gel extract, which was
stored at room temperature until ready for use.
Animal management and
grouping- Thirty-six (36)
adult male wistar rats weighing between 220 to 250 g were used for the study
and were kept in a plastic cage with iron nettings. They were placed in a well
ventilated standard room with twelve hours light and twelve hours darkness. The
animals were acclimatized for two weeks before the commencement of administration.
During this period, the animals were observed to ensure that they were disease-free
and were fed with rat chows, given water ad libitum, at an ambient temperature
range of 25–27 maintained at 50% humidity. The animals were weighed with an electronic weighing balance before the
commencement and termination of the administration of cannabis. Animals were
divided into four groups (A, B, C and D) with nine (9) rats in each.
Animal
Treatment/administration- Group A received normal saline,
group B received 200 mg/kg of cannabis, group C received 500 mg/kg, while group
D received 800 mg/kg of cannabis daily between 8.00–11.00 hours for fourteen
(14) consecutive days. Route of
administration was via oral.
Table 1: Animal grouping
and administration
Groups |
Dosage of cannabis administered |
Duration (Days) |
A |
Normal
saline |
14 |
B |
200
mg/kg |
14 |
C |
500
mg/kg |
14 |
D |
800
mg/kg |
14 |
Collection of Tissues- Twenty
four (24) hours after the last day of administration, the animals were weighed,
anesthetized with ketamine (80 mg/kg IP) and xylazine (11 mg/kg IP) and blood
sample was taken by cardiac puncture within seventeen (17) minutes. The testis
was then removed and preserved in 10% buffered formalin for 48 hours.
Biochemical
analysis (Hormonal Assay)- The collected blood was allowed to clot and
the serum put in a test tube and centrifuged at 300 rev per minute. The
supernatants were collected with the aid of a pasteur pipette as sample.
Ichroma quantitative testosterone method
(fluorescence immunoassay) was used to determine testosterone, follicle
stimulating hormone and Luteinizing hormone levels in serum [19].
Testosterone is used as an aid in the screening and monitoring of androgen
level.
Statistical Analysis- Statistical testing was
carried out using SPSS version 23, with P≤0.05 as
level of significance. Newman-Keuls Multiple
comparison test was used to compare measurability between experimental groups
and control group (one way ANOVA). Amscope 5.0 digital microscope was used to
make the micrographs after processing the testis in the histology laboratory.
Ethical
Approval- The study was carried
out by the principles of laboratory animal care and standard experimental
procedures. It was approved by The Ethical Committee of College of Medicine,
Enugu State University of Science and Technology, Enugu, Nigeria.
RESULTS- Table 2 shows how the consumption of C. sativa
affected the secretion of hormones across groups. The group administered with
800 mg/kg of C. sativa
had the lowest hormonal secretion level. Thus, cannabis extracts reduced testosterone,
FSH and LH levels.
Table
2: Showing
the distribution of effect of C. sativa
on the testicular hormones
|
Control |
200 mg/kg |
500 mg/kg |
800 mg/kg |
p-value |
Testosterone
(mµ/ml) |
2.16±0.08 |
1.82±0.10 |
1.48±0.10 |
1.23±0.03 |
0.00 |
LH
(mµ/ml) |
2.50±0.05 |
1.89±0.02 |
1.02±0.04 |
0.40±0.06 |
0.00 |
FSH
(mµ/ml) |
1.62±0.07 |
1.08±0.04 |
0.49±0.01 |
0.29±0.02 |
0.00 |
There was a decrease in levels of
testosterone, LH and FSH across experimental groups compared to the control
group. Not statistically significant. Fig. 1 shows that the graphical representation among the
relationship between the rate of Testosterone, FSH and LH levels secreted
across the groups given various doses of C. sativa
and comparing it to the control group. Hormonal levels reached a peak of
reduction at 800 mg/kg of C. sativa.
Fig.
1:
Comparison of hormonal assay among
groups in response to various doses of C. sativa
Serum testosterone,
FSH and LH decreased significantly among the experimental groups compared to
the control as doses increased (P<0.05), one-way ANOVA shown in Fig. 2.
Fig. 2: Testicular weight: Newman-Keuls Multiple
comparison test between experimental groups and control group with P≤0.05
(one way ANOVA)
There was a significant difference
between the weight of the testis between the control group and all the experimental
groups. Level of comparison of weight difference of testis between the control
and group D (800 mg/kg of C. sativa) was higher than groups B (200
mg/kg) and C (500 mg/kg). P=0.0085 (Statistically significant).
Histological
findings
DISCUSSION-
This study investigated the biochemical,
morphological and histological effect of C. sativa on the testis of albino wistar
rats. Testosterone, follicle-stimulating and luteinizing hormone levels were
checked. In this study, C. sativa
extract caused significant decrease in hormonal levels (p<0.05), when
compared with the control in all the hormones assayed, testosterone (2.16±0.08,
1.23±0.03), luteinizing hormone (2.50±0.05, 0.40±0.06) and follicle-stimulating
hormone (1.62±0.07, 0.29±0.02) especially at 800 mg/kg.
This may be due to the temporary impairment of cannabinoids in the pituitary
function reflected in decreased LH and FSH levels and hence reduced
testosterone levels. The decreased serum concentrations of testosterone and LH
in our findings, was consistent with results of findings from Elazeem et al. [11] and also concurs
with studies of Adu [15] and Kolodny [16]. The decrease
in testosterone could be attributed to inhibition of the gonadotrophin
releasing hormone in the hypothalamus by 9-tetrahydrocannabinol (THC) [17].
Thus, 800 mg/kg of C. sativa caused
significant decrease in hormonal level in serum.
C. sativa extracts also had
significant effects in the weight distribution of the testis of animals used
for this study. Fig. 2 revealed that the weight of the testis of animals in
group D (800 mg/kg) reduced drastically (SEM±3.1 g) against the weight of the
control group (SEM±5.85). That of group B (200 mg/kg) weighed an average of
±4.3, while that of group C (500 mg/kg) weighed an average of ±4.03. Payne and
his group of researchers conducted a research on effects on C. sativa extract on sperm parameters
and testis and also observed that there was slight decrease in testicular
weight of wistar rats used for the study [18].
Histological findings showed that 500 mg/kg and 800 mg/kg of C. sativa caused cellular damages in the spermatogenic area of the
seminiferous tubules of the testis of adult wistar rats as revealed by Fig. 5
and 6. Also, sertoli cell population was reduced in the germinal epithelium of
the seminiferous tubules as shown in Fig. 6 administered with 800 mg/kg of C. sativa. In the study of Mandal
and Nas [13], this was a prognosis of testicular regeneration
and is consistent with the histological findings of a
significant reduction in tubular diameter and detrimental changes in the
seminiferous epithelium of the testes, resulting in lower serum testosterone
and pituitary gonadotropin (LH and FSH) levels in the study.
CONCLUSIONS- The present research has shown
that C. sativa has an adverse effect on the serum concentration of gonadotrophic
hormones and also reduced sperm population in the germinal area of the
seminiferous tubules of the testis of adult wistar rats. This exposure of an
individual to C. sativa
could lead to a significant reduction of testosterone levels and subsequently
cause sertoli cells dysplasia. Thus, could lead to sub-fertility issue in a man. Also, testicular weight reduction in animals used for this
study indicates effects of ingestion of C. sativa extracts and finally corroborating that extract can has significant
effect on testicular weight, which constitutes part of the total body weight of
an individual.
Thus, can resultantly contribute to body weight loss, hence
more research should advocate screening of
gonadal hormones among acute drug abusers in addition to other metabolic and
psychiatric support. Further studies should also be carried out on any possible
effect of C. sativa on the interstitial cells
of the testis.
CONTRIBUTION OF AUTHORS
Research concept- Dr. Ignatius Ikemefuna Ozor
Research design- Dr. Ignatius Ikemefuna Ozor, Onyinye Mary
Ozioko, Uche Sebastine Ozioko, Christian Ejuiwa Mba, Dr. Ifeanacho Abireh,
Offor Chukwuebuka Blessed
Supervision- Dr. Ignatius Ikemefuna Ozor
Materials- Dr. Ignatius Ikemefuna Ozor,
Dr. Ifeanacho Abireh
Data collection- Dr. Ignatius Ikemefuna Ozor,
Christian Mba
Data analysis and Interpretation- Dr. Ignatius
Ikemefuna Ozor, Christian Mba
Literature search- Dr. Ignatius Ikemefuna Ozor,
Offor Chukwuebuka Blessed, Uche Ozioko
Writing article- Dr. Ignatius Ikemefuna Ozor, Onyinye Mary Ozioko,
Uche Sebastine Ozioko, Christian Ejuiwa Mba, Ifeanacho Ezeteonu Abireh, Offor
Chukwuebuka Blessed
Critical review- Dr. Ignatius Ikemefuna Ozor,
Dr. Ifeanacho Abireh
Article editing- Dr. Ignatius Ikemefuna Ozor,
Christian Mba, Ozioko Onyinye
Final approval- Dr. Ignatius Ikemefuna Ozor
REFERENCES
1.
Ruiz P, Strain
EC. Substance Abuse: A Comprehensive Textbook. Lippincott Williams &
Wilkins, 2011; pp. 214.
2.
Vij
K. Textbook of Forensic Medicine and Toxicology: Principles and Practice.
Elsevier India. 2011; pp. 672.
3.
Russo
EB. Cannabis and Cannabinoids: Pharmacology, Toxicology, and Therapeutic
Potential. Routledge, 2013; pp. 28.
4.
Newton
DE. Cannabis sativa: a reference handbook. Santa Barbara, Calif.:
ABC-CLIO. 2013; pp. 7; ISBN 9781610691499.
5.
Osborne GB, Fogel
C. Understanding the motivations for recreational cannabis sativa use
among adult Canadians. Substance
Use & Misuse, 2009; 43:
539–72. doi: 10.1080/10826080701884911.
6.
Riviello
RJ. Manual of forensic emergency medicine: a guide for clinicians.
Sudbury, Mass. Jones and Bartlett Publishers: 2010; pp. 41.
7.
D'Souza DC,
Sewell RA, Ranganathan M. Cannabis and psychosis/schizophrenia: human studies.
Eur Arch Psychiatry Clin Neurosci., 2009; 259 (7): 413–31. doi: 10.1007/s00406-009-0024-2.
8.
Crippa JA, Zuardi AW,
Martín-Santos R, Bhattacharyya S, Atakan Z, et al. Cannabis and anxiety: a
critical review of the evidence. Human
Psychopharmacol., 2009; 24(7):
515-23. doi: 10.1002/hup.1048.
9. Commonly used Drugs Charts: Cannabis sativa
(Cannabis). NIDA, US National Institutes of Health. 22 July, 2019, https://www.drugabuse.gov/drug-topics/commonly-used-drugs-charts.
10. Gordon AJ, Conley JW, Gordon JM. Medical consequences of Cannabis sativa use: a review of
current literature. Curr Psychiatry Rep., 2013; 15(12):
419. doi: 10.1007/s11920-013-0419-7.
11. Elazeem
RA, Modawe G, Abdelrahman SF. Assessment of serum luteinizing hormone, follicle-stimulating
hormone and testosterone level among some Sudanese people; Marijuana abuse
people. Sch Bull., 2015; 1(6): 148–50.
12. Alagbonsi IA, Olayaki LA. Ameliorative effect of combined
melatonin and vitamin C on Cannabis sativa-induced reproductive
hormonal toxicity. J Afr Assoc Physiol Sci., 2016;
4(1): 14–24.
13. Mandal
TK, Das NS. Testicular toxicity in cannabis extract treated mice: association
with oxidative stress and role of antioxidant enzyme systems. Toxicol Ind Health, 2010; 26(1):
11–23.
14. Berkow R, et al. The Merck manual of medical information (Home edition).
Merck Research Laboratories, 1997.
15. Adu
E, Popoola O, Adikema N. Serum testosterone level in Nigerian Cannabis sativa and cigarette
smokers. Afr. J. Cell Path.,
2014; 2(3): 35–39.
16. Kolodny
RC, Masters WH, Kolodner RM, Toro G. Depression of plasma testosterone levels
after chronic intensive marihuana use. N Eng J Med., 1974; 290(16):
872–74.
17. Meccariello
R, Chianese R, Fasano S, Pierantoni R. Endocannabinoids and kisspeptins: two
modulators in fight for the regulation of GnRH activity. Gonadotropin IntechOpen., London,
UK,2013.
18. Payne
KS, Mazur DJ, Hotaling JM, PastuszakAW. Cannabis and male fertility: A
systematic review. J Urol., 2019; 202(4): 674–81. doi:
10.1097/ju.0000000000000248.
19. Bolodeoku
J. The Accuracy Performance of the Point of Care Test (POCT) Boditech I-CHROMA™
Testosterone Method using External Quality Assessment Schemes: RIQAS and
UKNEQAS. Am J Bio Sci Res., 2019; 4(6): 000859.