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American Journal of Research Communication www.usa-journals.com
Akunna, et al., 2015: Vol 3(1) ajrc.journal@gmail.com
Histo-morphometric Evidences for Testicular Derangement in animal
models submitted to chronic and Sub-chronic Inhalation of Fragrance
1*Akunna G.G.; 1Saalu L.C. 2Ogunlade B.; 1Akingbade A.M.; 3Anderson L.E.;
4Olusolade F.S
1 Department of Anatomy, College of Medicine, Lagos State University, Ikeja Lagos, Nigeria
2 Department of Anatomy, College of Medicine, University of Lagos, Idi Araba, Lagos,
Nigeria
3 Department of Anatomy, Afe Babalola University, Ado Ekiti, Nigeria
4 Department of Parasitology, Afe Babalola University, Ado Ekiti, Nigeria
Corresponding Author: AKUNNA, Gabriel Godson
Department of Anatomy, College of Medicine, Lagos State University, Ikeja Lagos, Nigeria
+2348038619526, ggakunna@yahoo.com
ABSTRACT
Only few people are aware that in the manufacturing of a single bottle of perfume, about 600
individual chemical ingredients coined as fragrance are used. Exposure and a lingering
culture of trade secrecy and undisclosed substances within the fragrance industry has
remained an important factor when considering the hazardous and degenerative effect of
these so called scents.
In this study, we evaluated the histopathological effect of two popular perfumes used in
Nigeria on the testis of rat. Sixty adult male Wistar rats were randomly divided into six
groups of ten rats each. Group A and B rats (Controls) rats were exposed (6hrs day-1) to 5 ml
kg-1 body weight of normal saline for 56 days and 112 days via whole body inhalation
respectively, Group C and D rats were exposed (6hrs day-1) to 5 ml kg-1 body weight of one
of the perfume designated as F1 for a period of 56 days and 112 days via whole body
inhalation respectively while Group E and Group F rats were exposed (6hrs day-1) to 5 ml kg-
1 body weight of one of the perfume designated as F2 for a period of 56 days and 112 days via
whole body inhalation respectively.
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Result indicated that the testes of exposed groups of rat had worst geometric values and
histological profiles compared to the control group of rat. These results indicated and
validated the histopathological role of fragrance components in rat.
Keywords: Fragrance, Testes, Stereology, Infertility, Oxidative Stress
{Citation: Akunna G.G., Saalu L.C., Ogunlade B., Akingbade A.M., Anderson L.E.,
Olusolade F.S. Histo-morphometric evidences for testicular derangement in animal models
submitted to chronic and sub-chronic inhalation of fragrance. American Journal of Research
Communication, 2015, 3(1):} www.usa-journals.com, ISSN: 2325-4076.
INTRODUCTION
A sturdy debate over whether male reproductive ability is influenced by environmental
factors has long persisted. In 1977, there was a remarkable and sensational report by Whorton
et al. (1977) in which 14 out of 25 male workers involved in producing dibromo-3-
chloropropane (DBCP), were diagnosed as azoospermic or oligospermic. In 1992, Carlsen et
al. (1992) reported a marked decrease in sperm count in previous 50 years. While some
researchers continue registering their doubts over any correlation between environmental
factors and male infertility (Chapin et al., 1994), many researchers have asserted that societal
progress in advanced countries and worsening of the natural environment have an imperative
negative bearing on male fertility. Long-reported risk factors include exposure to radiation,
electromagnetic waves, and a variety of chemical substances including fragrance.
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Fragrance refers to a combination of substances that gives perfume or cologne a unique scent.
Copious documentations have indicated that 82 percent of perfumes labeled “natural
ingredients” actually contain synthetic fragrances (Rastogi et al., 1996).
The consumers are kept back about the actual chemicals in fragrance that poses a potential
health risks. Such chemicals that affect male reproductive hormones may be a factor in
infertility and has been known as endocrine disruptors (Giudice, 2006, Saalu et al., 2010,
Akunna et al., 2013).
It has been reported that perfumes, colognes, body sprays and care products contained an
average of four potential hormone-disrupting chemicals.
In male reproductive anatomy, endocrine disruptors have severally been implicated as
teratogens, resulting in cryptorchidism, hypospadias and impairment of body function
normally regulated by natural hormone signaling (Wang and Baskin, 2008, Akunna et al.,
2011, Akunna et al., 2013). Studies have shown that these chemicals causes damage by
mimicking or disrupting natural estrogen, testosterone and thyroid pathways (Soto et al.,
2009).
Although the implication of subsequent exposure to these chemicals have not been critically
understood, recent findings has clearly demonstrated disruption in spermatogenesis (Akunna
et al., 2014), liver damage (Akunna et al., 2011) and other tissue toxicity in animals exposed
to fragrance components (Johansen et al., 2003, Elberling et al., 2004, Breast Cancer Fund,
2008, Schnuch et al., 2010). In animal model studies, fragrance exposure has lead to
spermatotoxicity and infertility, congenital malformation in penises and abnormal testes
(Akunna et al., 2014).
According to published scientific studies, diethyl phthalate and octinoxate which are major
components of perfume and sunscreen respectively has been implicated in sperm damage,
apoptosis and interference with estrogen and androgens in human respectively (Giudice,
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2006, Wang and Baskin, 2008, Silva et al., 2004 ,Schreurs et al., 2005, Swan, 2008, CDC,
2009). Sperm DNA damage was reported in a study of 168 men and 379 men exposed to
diethyl phthalate and DEHP respectively (Duty et al., 2003, Hauser et al., 2007, López-
Carrillo et al., 2010).
A strong relationship between diethyl phthalate exposure during pregnancy and changes in
male genital development and alterations in levels of male sex hormones in the baby boys has
been established (Swan et al., 2005, Main et al., 2006).
However, some animal model studies of fragrance components could not report any alteration
in the reproductive anatomy (Howdeshell et al., 2008). However, at the highest levels of
exposure, DEP has been linked to liver abnormalities, elevated cholesterol (Sonde et al.,
2000) and birth defects (ATSDR, 1995). In our laboratory in 2013, we reported a decrease in
sperm count, motility and an increase in abnormal sperm morphology following exposure to
fragrance components (Akunna et al., 2011, Kwack et al., 2009). In this study, we
demonstrated the testicular histopathological effect of these components in rat using
scientifically proven histo-morphometric principles.
MATERIALS AND METHODS
In this study, two (2) commonly used perfumes in Nigeria designated as F1 and F2 were
obtained from Bayous Cosmetics in Lagos, Nigeria and were kept under standard
temperature. As described by Akunna et al. (2011), sixty adult male Wistar rat (12-13 weeks
old) weighing 190-220 g were used for the study. The rats were randomly divided into six
groups (A-F) of ten rats and the average weight difference between and within groups did not
exceed ± 20% of the average weight of the sample population. Group A rats served as the
first control (Control I) and were exposed (6hrs day-1) to 5 ml kg-1 body weight of normal
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saline for 56 days. Group B rats served as the second control (Control II) group and were
exposed (6hrs day-1) to 5 ml kg-1 body weight of normal saline for a period of 112 days.
Group C (Sub-chronic I) and Group D (Chronic I) rats were exposed (6hrs day-1) to 5 ml kg-
1 body weight of Fragrance I (F1) for a period of 56 days and 112 days respectively. Group E
(Sub-chronic II) and Group F (Chronic II) animals were exposed (6hrs day-1) to 5 ml kg-1
body weight of Fragrance II (F2) for a period of 56 days and 112 days respectively. The study
is consistent with the standard of the use of laboratory animals. Small balls of cotton wool
were soaked with the fragrance (Experimental Groups) and normal saline (Control Groups)
respectively. The wools were then placed in a Petri dish inside the cages and covered with
perforated plastic to prevent direct contact for an exposed duration of 6hrs day-1 throughout
the period of study (Akunna et al., 2011).
Animal sacrifice and sample collection
The rats were first weighed and then were sacrificed by cervical dislocation. The abdominal
cavity was opened up through a midline abdominal incision to expose the reproductive
organs. The testes were excised and trimmed of all fat. The testicular weights of each animal
were evaluated with an electronic analytical and precision balance (BA 210S, d=0.0001-
Sartoriusen GA, Goettingen, Germany). The testes volumes were measured by water
displacement method. The two testes of each rat were measured and the average value
obtained for each of the two parameters was regarded as one observation. One of the testes of
each animal was fixed in 10% formol-saline for histological examination (Akunna et al.,
2013).
Histo-morphometric evaluation
The testes after whole body perfusions were transferred to a graded series of ethanol. On day
1, they were placed in 70% alcohol for 7 hours, then transferred to 90% alcohol and left in the
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latter overnight. On day 2, the tissues were passed through three changes of absolute alcohol
for an hour each and then cleared in xylene. Once cleared, the tissues were infiltrated in
molten paraffin wax in the oven at 58ºC.
Three changes of molten paraffin wax at one-hour interval were made, after which the tissues
were embedded in wax and blocked out. Prior to embedding, it was ensured that the mounted
sections to be cut by the rotary microtome were orientated perpendicular to the long axes of
the testes. These sections were designated “vertical sections”. Serial sections of 5 μm thick
were obtained from a solid block of tissue, fixed on clean slides to which Mayer’s egg
albumin had been coated to cement the sections to the slides properly and were stained with
haematoxylin and eosin stains, after which they were passed through a mixture of equal
concentration of xylene and alcohol. Following clearance in xylene the sections were oven-
dried between 35°C and 40°C (Sheehan and Hrapchak, 1987).
The slides were viewed under a research microscope connected to a computer monitor for
qualitative and quantitative evaluation. For each testis, seven “vertical sections” from the
polar and the equatorial regions were sampled (Gundersen and Jenson, 1987) and an unbiased
numerical estimation of the following morphometric parameters was determined using a
systematic random scheme: testicular volume and weight; diameter (D) and cross-sectional
area of the seminiferous tubules (AC); number of profiles of seminiferous tubules per unit
area of testis (NA); and numerical density of the seminiferous tubules (NV). Seven “vertical
sections” per testis were selected by a systematic sampling method that ensured fair
distribution between the polar and equatorial regions of each testis. Briefly, a section was
taken at the equator of each testis; one on each side of the equator, three-quarters of the
distance between the pole and the equator; another half-way between each pole and the
equator; and one on each side of the equator, a quarter of the distance from each of the pole.
For each stereological parameter (D, AC, NA and NV), five randomly selected fields from all
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the seven sections of a single testis were viewed, and estimation on each carried out. The
average from a total of seventy readings from five fields in seven sections of the two testes of
one rat was obtained and this was recorded as one observation. The evaluation of the
diameter was done with calibrated eyepiece and stage grids mounted on a light research
microscope at X 100 magnification. Estimation of volume density of testicular components
and number of seminiferous tubules were done on a computer monitor onto which a graph
sheet was superimposed and on which slides were projected from a research light
microscope.
Diameter (D) of seminiferous tubules: The diameter of seminiferous tubules with profiles
that were round or nearly round were measured for each animal and a mean, D, was
determined by taking the average of two diameters, D1and D2 (Perpendicular to one another).
D1and D2were taken only when D1/D2 ≥ 0.85.
Cross-sectional area (AC) of the seminiferous tubules: The cross-sectional areas of the
seminiferous tubules were determined from the formula AC = πD2/4, (where π is equivalent to
3.142 and D the mean diameter of the seminiferous tubules).
Number of profiles of seminiferous tubules in a unit areaof testis (NA): The Number of
profiles of seminiferous tubules per unit area was determined by using the unbiased counting
frame proposed by Gundersen and Jenson (1987). Using this frame, in addition to counting
profiles completely inside the frame we counted all profiles with any part inside the frame
provided they did not touch or intersect the forbidden line (full-drawn line) or exclusion
edges or their extension.
Numerical Density (NV) of seminiferous tubules: This is the number of profiles per unit
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volume and was determined by using the modified Floderus equation:
NV =NA/ (D +T) (Gundersen and Jenson, 1987) where, NA is the number of profiles per unit
area, D is the diameter and T the average thickness of the section.
Statistical Analysis
All data were expressed as mean ± SD of number of experiments (n = 10). The level of
homogeneity among the groups was tested using Analysis of Variance (ANOVA) as done by
(Snedecor and Cochran, 1980). Where heterogeneity occurred, the groups were separated using
Duncan Multiple Range Test (DMRT). Values of p < 0.05 and P < 0.005 were considered to
indicate a significant difference between groups (Duncan, 1957). Analysis of data was done
using both electronic calculator and Statistical Package for Social Sciences (SPSS)/ PC
computer program (version 11.0 SPSS, Cary, NC, USA).
RESULT AND DISCUSSION
Changes in gross anatomical parameters
There was a significant decrease (p<0.05 and p<0.005 at 56days and 112days respectively)
in body weight, testicular weight, testicular volume and testis weight/body weight ratio of
exposed rats which was time dependent. This could be due to reduction in food intake by the
exposed animals and the level of stress which could have been caused in part, by the inhaled
substances (Akunna et al., 2011). The result in this study conform to previous reports of
significant reduction in gross anatomical parameters due loss in testicular integrity as a result
of various oxidative derangements (Akunna et al., 2011, 2014). Carlssen et al. (1992)
reported a decrease in body weight and gonad somatic index following exposure of animal
models to musk ketone.
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The significant decrease in testicular weight in our study could have been as a result of
Sertoli cell apoptosis due to fragrance toxicity. A direct association between testis size and
spermatogenesis has been documented. Testicular size which is said to be determined at
perinatal and prepubertal period when the sertoli cell mass is established. More Sertoli cells
means more germ cells per testis, and the number of sertoli cells per gram of tissue combined
with the number of spermatids per sertoli cells is associated with sperm production per gram
of testis (Saalu et al., 2008). This report is in accordance with our previous report on
decreased sperm concentration, sperm motility and increase in abnormal sperm morphology
of rat exposed to fragrance. As shown in Table [1], the control group of rats had a significant
(p<0.005) increase in body weight when compared to fragrance-exposed rats that loss
significant amount of weight. Again the increase in body weight of the control rats could
mean that they were still in their active growth phase during the study (Saalu et al., 2008,
2011).
Table 1: Body weight changes in experimental animal exposed (6hrs day-1) to fragrance
components (5ml/kg body weight for 56 days and 112 days). Values are expressed as
Mean ± SD for n=10; P*<0.05, P**<0.005 significantly different from control.
Gross
Anatomical
Parameters
Initial Body
Weight
(g)
Final Body
Weight
(g)
Body
Weight.
Differences
Group A
220.3±0.1
273.1±3.1
52.8
Group B
210.2±1.4
281.1±1.4
70.9
Group C
205.6±0.1
180±1.3
25.6*
Group D
198±1.1
140±0.4
58*
Group E
Group F
210.1±2.2
203.1±5.0
180±3.2
150.3±1.0
30.1**
52.8**
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Where:
A: 5 ml kg-1 body weight normal saline (6hrs day-1) for 56 days (Control I)
B: 5 ml kg-1 body weight of normal saline (6hrs day-1) for 112 days (Control II)
C: 5 ml kg-1 body weight of F1 (6hrs day-1) for 56 days (Sub-chronic I)
D: 5 ml kg-1 body weight of F1 (6hrs day-1) for 112 days (chronic I)
E: 5 ml kg-1 body weight of F2 (6hrs day-1) for 56 days (Sub-chronic II)
F: 5 ml kg-1 body weight of F2 (6hrs day-1) for 112 days (Chronic II)
Figure 1: The effect of Fragrance exposure (5ml kg-1 body weight for 56 days and 112
days) on testicular weight, testicular volume and testicular weight/body weight ratio of
experimental rat. Values are expressed as Mean ± SD for n=10; P*<0.05, P**<0.005
significantly different from control.
Where:
A: 5 ml kg-1 body weight normal saline (6hrs day-1) for 56 days (Control I)
B: 5 ml kg-1 body weight of normal saline (6hrs day-1) for 112 days (Control II)
C: 5 ml kg-1 body weight of F1 (6hrs day-1) for 56 days (Sub-chronic I)
D: 5 ml kg-1 body weight of F1 (6hrs day-1) for 112 days (chronic I)
E: 5 ml kg-1 body weight of F2 (6hrs day-1) for 56 days (Sub-chronic II)
F: 5 ml kg-1 body weight of F2 (6hrs day-1) for 112 days (Chronic II)
Testicular morphometry
In other to quantify the histological changes in this study scientifically, stereological
evaluation of the tissues was employed. It has been shown that the level of testicular
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androgen is directly proportional to the size of the testicular interstitium and the number of
Leydig cell (Yama et al., 2011, Akunna et al., 2014).
In this study, there was a significant (p<0.05 at 56days and 112days respectively)
reduction in the mean seminiferous tubular diameters, cross sectional area, number of profiles
per unit area and the mean numerical density of seminiferous tubules of rat exposed to
fragrance components (5 ml kg-1 b. wt for 56 days and 112 days) when compared to the
control groups that had significant increase in stereological parameters [Table 2].
In experimental animals such as rats, the interstitial and tubular compartment comprises
about 2.6% and about 60–80% of the total testicular volume. In the human testis, the
interstitial compartment represents about 12–15% of the total testicular volume, 10–20% of
which is occupied by Leydig cells. The Leydig cells being the most important cell of the
interstitium and the source of testicular testosterone and of insulin-like factor 3 (INSL3).
Deducing from previous documentation, our result herein could only suggest a
significant decrease in interstitium which could be due to reduction in the number of
androgen producing cells (Interstitial cells of Leydig) (Saalu et al., 2006, Yama et al., 2011).
However, the stereological changes evidenced in our study were not time-dependent.
Although the stereological values obtained from our study are actually sound evidence
of the three-dimensional characteristics of the rat testis exposed to fragrance components, it
will be scientifically incorrect to predict the expected consequence of these degenerative
changes on entire spermatogenic process because the present morphometric data only forms
part of the entire delineation. Other factors are clearly important. These include number of
spermatogenic cells in the basal compartment and the Sertoli-Sertoli cell barrier which
determines the number of cells in the adluminal compartment (Yama et al., 2011).
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Table 2: The effect of Fragrance components (5 ml kg-1 b. wt for 56 days and 112 days)
on seminiferous tubular diameter (μm), cross sectional area Ac (×103μm2), numerical
densities of seminiferous tubules NA (×10-8μm-2) and number of profiles per unit area
Nv (×10-10μm-3). Values are expressed as Mean ± SD for n=10; P*<0.05, P**<0.005
significantly different from control.
Treatment Groups
D(μm)
Ac (×103μm2)
NA (×10-8μm-2)
Nv (×10-10μm-3)
Group A
161.3±2.3
33±1.1
29.1±1.8
21.3±2.2
Group B
152±8.1
34±1.2
30.2±1.1
20.1±2.1
Group C
110±2.2*
26.1±0.1*
20.2±3.3*
15±1.3*
Group D
107.3±0.3*
20±1.1*
14.1±3.1*
12.3±1.6*
Group E
Group F
125.9±0.3*
96.3±2.0*
24.8±3.3*
22±0.4*
20.2±2.1*
17.3±9.0*
17.1±2.1*
13.1±4.0*
Where:
A: 5 ml kg-1 body weight normal saline (6hrs day-1) for 56 days (Control I)
B: 5 ml kg-1 body weight of normal saline (6hrs day-1) for 112 days (Control II)
C: 5 ml kg-1 body weight of F1 (6hrs day-1) for 56 days (Sub-chronic I)
D: 5 ml kg-1 body weight of F1 (6hrs day-1) for 112 days (chronic I)
E: 5 ml kg-1 body weight of F2 (6hrs day-1) for 56 days (Sub-chronic II)
F: 5 ml kg-1 body weight of F2 (6hrs day-1) for 112 days (Chronic II)
Histological profile of the testicular tissue
Increased spermatogenic efficiency has been implicated with higher seminiferous epithelium,
increased number of spermatogonia production, and decreased germ cell loss (Saalu et al.,
2007).
About 35–40% of the volume of the germinal epithelium is represented by Sertoli cells. The
intact testis with complete spermatogenesis contains 800–1200 × 106 Sertoli cells (Zhengwei
et al., 1998) or approximately 25 × 106 Sertoli cells per gram testis (Raleigh et al., 2004).
In this study, rats that were exposed to fragrance showed destructive changes in their
seminiferous tubular epithelium and interstitial tissues evidenced by an uneven arrangement
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at the basal portion of the germinal epithelium Fig [4-7]. There profile was characterized by
hypo spermatozoa formation in the tubules when compared to the control group of rat Fig. [2-
3]. The tubular epithelium of groups of rat treated for 54 days (Sub-chronic exposure)
exhibited lesser pathological alterations to the when compared to the group treated for 112
days (Chronic exposure). This was a sound conclusion of the time dependent manner of
fragrance toxicity.
From our studies on fragrance, we can conclude herein that fragrance components are
testiculotoxic in rat. Although numerous reports on the effect of fragrance on human health
have been documented, there is a need for further investigation on whether fragrance acts
directly as spermatotoxins or through a steroidal pathway. This is more pertinent when one
consider that in species such as rat with lower proportion of Sertoli cells in the seminiferous
epithelium, there is higher Sertoli cell and greater spermatogenic efficiencies when compared
to humans.
Figure 2: Testicular histological profile of Group A rats (Control I) (5 ml kg-1 body
weight of normal saline for 56 days).
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Figure 3: Testicular histological profile of Group B rats (Control II) 5 ml kg-1 body
weight of normal saline for 112 days).
Figure 4: Testicular histological profile of Group C rats (5 ml kg-1 body weight of F1 for
56 days).
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Figure 5: Testicular histological profile of Group D rats (5 ml kg-1 body weight of F2 for
112 days).
Figure 6: Testicular histological profile of Group E rats (5 ml kg-1 body weight of F2) for
56 days).
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Figure 7: Testicular histological profile of Group E rats (5 ml kg-1 body weight of F2 for
112 days).
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