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Incense smoke inhalation affects spermatogenesis
and sperm quality and impairs liver and kidney
function in adult male rats
Tarek S. El Sewedy, Nadia F. Ismail, and Samah E. Ismail
INTERNATIONAL JOURNAL OF CANCER AND BIOMEDICAL RESEARCH (IJCBR)
RESEARCH ARTICLE
IJCBR (jcbr.journals.ekb.eg) is published by Egyptian Society of Cancer Research (eacr.tanta.edu.eg) and sponsored by the Egyptian Knowledge Bank (www.ekb.eg)
Incense smoke inhalation affects spermatogenesis and sperm
quality and impairs liver and kidney function in adult male rats
Tarek S. El Sewedy1, Nadia F. Ismail2, and Samah E. Ismail1
1Department of Applied Medical Chemistry, Medical Research Institute, Alexandria University, Alexandria, Egypt
2Department of Biochemistry, Faculty of Health Sciences Technology, Borg El Arab Technological University, Alexandria, Egypt
Background: Despite the growing evidence indicating the association between
inhalation of traditional incense smoke (IS) and the increased risk of numerous
health issues, its possible effect on spermatogenesis is still confusing. Aim: The
present study was designed to address the hypothesis that exposure to traditional
IS may impact sperm quality and affect liver and kidney function. Material and
Methods: Using a rat model, we evaluated the effects of IS exposure on semen
quality by Computer-Assisted Semen Analysis (CASA) and blood samples were
collected for the determination of random blood glucose, C-reactive protein (CRP),
liver and kidney function parameters and testosterone levels. Results: Prolonged
inhalation of IS caused a highly significant decrease in testosterone levels compared
with the control group. Moreover, complete semen analysis indicated that sperm’s
progressive motility, velocity, and vigor parameters were significantly decreased in
IS-exposed groups compared with the unexposed group. Also, IS inhalation
significantly increased the percentage of morphologically abnormal sperms.
Biochemically, prolonged inhalation of IS caused a significant decrease in blood
glucose while blood CRP, liver, and kidney function biomarkers were significantly
elevated compared with the control group, reflecting potential toxicity. Conclusion:
We concluded that excessive and prolonged IS inhalation may cause a remarkable
deterioration in spermatogenesis, sperm quality as well as kidney and liver function.
Keywords: Lincense smoke; Liver and kidney functions, Spermatogenesis
Editor-in-Chief: Prof. M.L. Salem, PhD - Article DOI: 10.21608/JCBR.2023.200215.1296
Article history
Received: March 15, 2023
Revised: April 25, 2023
Accepted: April 29, 2023
Correspondence to
Samah E. Ismail,
Department of Applied Medical
Chemistry, Medical Research Institute,
Alexandria University, Alexandria,
Egypt
Email: Samah.diab@alexu.edu.eg
Copyright
©2023 Tarek S. El Sewedy, Nadia F.
Ismail and Samah E. Ismail. This is an
Open Access article distributed under
the Creative Commons Attribution
License, which permits unrestricted
use, distribution, and reproduction in
any format provided that the original
work is properly cited.
INTRODUCTION
Incense (from Latin incendere “to burn”) is
made of aromatic biological materials and
essential oils when burn emitting fragrant
smoke (Qin et al., 2019). There are different
forms of incense available commercially
including sticks, cones, coils, rope, and smudge
bundles. It has been hypothesized that incense
burning is a source of indoor air pollution that
harms those who are exposed to it (Cohen et al.,
2013). Most types of incense sticks share the
same structure and various reports showed that
in general during their burning process, they
generate smoke consisting of a gaseous phase
of carbon dioxide, carbon monoxide, sulfur
dioxide, formaldehyde, nitrogen dioxide,
polycyclic aromatic compounds, volatile organic
compounds (VOCs), toxic heavy metals and
particulate matter (PM) (Friborg et al., 2008; Ji
et al., 2010; Geng et al., 2019; Yadav et al., 2020;
Yadav et al., 2021).
Several studies have reported elevated levels of
these toxic organic compounds in IS and
suggested a negative association with sperm
quality and the spermatogenesis process in
human and animal models (Xiao et al., 2001;
Yang et al., 2007; Zhang et al., 2015; Mahgoub
& Salih, 2017; Lv et al., 2022). Burning incense
at home is a long-standing and widespread
practice among the people of Asian nations and
the Middle East; these areas have higher
percentages of male infertility worldwide. A
minimum of 30 million men are diagnosed as
infertile worldwide, with the highest rates
detected in Africa (Agarwal et al., 2015). Male
infertility is generally owed to the decline in
counts and quality of spermatozoa which were
reported to be highly disrupted by oxidative
stress resulting from the generation of reactive
El Sewedy et al., 2023
IJCBR Vol. 7(2): 35-45
36
oxygen species (ROS) targeting spermatozoa
plasma membrane and DNA (Alahmar, 2019).
Exposure to environmental pollutants is
being recognized as a potential cause of
infertility and despite its worldwide increasing
incidence, limited reports focused on exploring
novel lifestyle-associated environmental risk
factors that might contribute to idiopathic male
infertility. (Gabrielsen & Tanrikut, 2016; Vecoli
et al., 2016; Jurewicz et al., 2018; Mima et al.,
2018) Growing evidence suggests an association
between exposure to IS and the increased risk
of numerous human health issues (Kim et al.,
2014; Kim et al., 2019; Akingbade et al., 2021;
Lee et al., 2021). Recently, it was demonstrated
that Carlina gummifera IS inhalation caused
oxidative stress in the testis, which may be the
indirect cause of the observed changes in
spermatogenesis (Dorsaf et al., 2022).
Therefore, the present study was designed to
investigate the hypothesis that prolonged IS
inhalation may damage sperm quality, liver and
kidney normal functions in adult male rats
subjected to low and high doses of IS.
MATERIAL AND METHODS
Animals
This study was conducted on forty-five sexually
mature Sprague-Dawley male rats, aged 70-80
days and weighing 130 – 150 g. Rats were
purchased and housed in the experimental
animal house, Faculty of Pharmacy, Pharos
University, Alexandria, Egypt. Rats were housed
in metal cages (15 rats/cage) and maintained at
approximately 23-25˚C with a 12:12h light/dark
cycle and received laboratory basal diet and tap
water for a one-week acclimation period and
throughout the whole study. All animal
procedures and experimental protocols were
approved by the Research Ethics Committee of
the medical research institute, Alexandria
University (AU0122282233). All animal
experiments were performed according to the
ARRIVE guidelines and the National Research
Council’s guide for the care and use of
laboratory animals.
Materials
The most common incense sticks in Egypt were
used in the current study (Ansam®,
0.4 g/Makkah incense stick, SAK).
Animal Treatments
Rats were randomly divided into three groups
(15 rats/ group). Group I included 15 rats
unexposed to IS and served as the control
group. Group II included 15 rats exposed to a
low dose of IS (1.6 g) for 30 min daily for four
weeks. Group III included 15 rats exposed to a
high dose of IS (3.2 g) for 30 min daily for four
weeks. Rats were exposed to incense smoke in
an inhalation chamber with dimensions of 55
cm by length, 40 cm by width, and 20 cm by
height as shown in Figure 1. The ratio of
chamber volume (44-L) to the mean body
weight of rats (140 g) was 314, which is in
proportionate to that of the ratio (317) of
standard male body weight (85 kg) in a standard
living room volume (27,000 L) (Yamamoto et al.,
2021).
Blood sample collection
Rats were sacrificed at the end of the
experiment and blood samples were taken
through heart puncture. The blood sample was
collected in a non-heparinized (plain) tube, left
to clot for 15 min and the serum was separated
by centrifugation then the serum was separated
and stored at -20 ◦C for further use.
Determination of Biochemical Parameters
Serum levels of glucose, alanine
aminotransferase (ALT), aspartate
aminotransferase (AST), urea, and creatinine
were determined according to manufacturer's
protocols (Bio-diagnostic Co., Egypt) using
double beam spectrophotometer (Shimadzu,
Kyoto Japan). Serum CRP and testosterone
levels were determined by enzyme-linked
immunosorbent assay (ELISA) according to the
manufacturer's protocols (DRG Diagnostics,
Marburg, Germany).
Figure 1. Incense smoke inhalation chamber
Incense smoke inhalation affects spermatogenesis and sperm quality and impairs liver and kidney function in adult male rats
IJCBR Vol. 7(2): 35-45
37
Semen Analysis
To allow spermatozoa release, freshly dissected
portions of the epididymis cauda were cut three
times and placed in a petri dish containing 500
µL tris-citric acid-fructose (Tris 3.025 g, citric
acid 1.7 g, fructose 1.25 g, distilled water 100
mL) and warmed for 5 minutes at 37 C. This fluid
was collected in aliquots for semen analysis
(Lima et al., 2018).
Computer-assisted semen analysis (CASA) with
a phase contrast microscope was used to assess
sperm motility immediately after collection. The
equipment was calibrated for rodent
spermatozoa, and motility parameters including
the spermatozoa motility (motility, %), the
progressive motility (PROG, %), the velocity
average path (VAP, m/s), the velocity curved
line (VCL, m/s), the velocity straight line (VSL,
m/s), wobble (WOB, %), straightness (STR, %),
linearity (LIN, %), beat cross frequency (BCF, Hz)
and amplitude of lateral head displacement
(ALH, m) were assessed.
For sperm morphology analysis, 50 µL of
epididymal fluid was fixed in 100 µL of buffered
formaldehyde (4%). 200 cells from each rat
were examined under phase-contrast
microscopy, and categorized as normal or
abnormal according to the strict sperm
morphology criteria. The morphological
abnormalities were divided into head, neck, and
tail defects. The percentages of normal and
abnormal-shaped sperms were determined.
Statistical analysis
Data were examined and analyzed using IBM
SPSS software package version 20.0. The
Kolmogorov-Smirnov test was used to verify the
normality of the distribution of quantitative
variables. Quantitative data were described
using range (minimum and maximum) and
mean ± standard deviation (SD). One-way
analysis of variance (ANOVA) was used for
normally distributed quantitative variables to
compare between more than two groups, and
the Post Hoc test (Bonferroni) for pairwise
comparisons. The significance of the obtained
results was judged at the 5% level. *P value ≤
0.05, **P ≤ 0.01, and ***P ≤ 0.001 were
considered significant, very significant, and
highly significant, respectively.
RESULTS
Effect of IS on testosterone levels
The range and mean ± SD for testosterone levels
(nmol/L) are shown in Table 1. Inhalation of IS
has caused a significant decrease in
testosterone levels in low-dose and high-dose
groups compared to the control unexposed
group (0.28 ± 0.03, 0.3 ± 0.1, and 3.9 ± 0.2,
respectively).
Effect of IS on sperm quality
As presented in Table 2, the motile spermatozoa
(MOT, %), in low and high-dose groups were
significantly higher than that in the control
unexposed group (P= 0.008, p= 0.001;
respectively). However, sperm progressive
motility (PROG, %) in low and high dose groups
was lower in low and high dose groups
compared to the unexposed group (12.8 ± 0.4,
12.8 ± 1.2, and 15.3 ± 0.5, respectively).
Moreover, the mean sperm velocity average
path (VAP, m/s), velocity curved line (VCL, m/s)
and velocity straight line VSL (m/s) in IS exposed
groups were significantly lower than the control
group. Additionally, the mean wobble (WOB, %)
in low-dose and high-dose groups were
significantly lower than that in the unexposed
group (34.3 ± 2.99, 39.2 ± 5, and 50 ± 3.8,
respectively). On the other hand, statistically
insignificant decreases in straightness (STR, %),
linearity (LIN, %), the amplitude of lateral head
displacement (ALH, m) and beat cross frequency
(BCF, Hz) were observed in IS-exposed groups
compared to the controls. Sperm morphology
analysis is shown in Table 3, the sperm
deformity index (SDI) value in low-dose and
high-dose groups was significantly higher than
that in the unexposed group (p=0.003, p=0.02;
respectively). The mean (%) of normal sperms in
low and high-dose groups was significantly
lower than that in the control unexposed group
(9.6 ± 2.4, 9 ± 2.5, and 14.15 ± 0.1, respectively).
Finally, the tera to sperm percentage (sperms
with abnormal morphology) in low-dose and
high-dose groups was significantly higher than
that in the unexposed group (90.4 ± 2.4, 91 ±
2.5, and 85.8 ± 0.1, respectively). The
predominant morphological defects were
tapered or big heads, small acrosome heads,
bent or thick necks, asymmetric insertion necks,
bent, broken, or coiled tails.
El Sewedy et al., 2023
IJCBR Vol. 7(2): 35-45
38
Figure 2. Microphotographs illustrating sperm morphology in the unexposed group (IA & IB), low-dose group (IIA-IID), and
high-dose group (IIIA & IIIB).
Incense smoke inhalation affects spermatogenesis and sperm quality and impairs liver and kidney function in adult male rats
IJCBR Vol. 7(2): 35-45
39
Table 1. Testosterone levels in studied groups.
Testosterone level (nmol/L)
Group I (n = 15)!
Group II (n = 15)!
Group III (n = 15)!
F!
p!
Range
"#$!%!&#'!
(#'!%!(#"!
(#)*!%!(#&!
)+$)#$,)*!
-(#(()*!
Mean ± SD
"#,!.!(#'!
(#'/!.!(#("!
(#"!.!(#)!
Significance
01-(#(()***1!02-(#(()***!032(#/+*!
34560 I: unexposed, Group II: low-dose group, and Group III: high-dose group. p1: p value for comparing between group I and group II, p2: p
value for comparing between group I and group III, p3: p value for comparing between group II and group III ***: Statistically significant at p
≤ 0.001.
Table 2. Sperm motility analysis
!
!
Group I (n = 15)!
Group II (n = 15)!
Group III (n = 15)!
F!
P!
MOT (%)
789:;!
'+#/!<!'$#"!
'*#,,!%!"(#"!
',#,!<!")#)!
'$#+!
(#(()!
=;89!.!>?!
'$#(&.(#'!
',#)!.!)#'!
"(#+!.!(#$!
>@:9@A@B89B;!
C)2(#((/**1!C'2(#(()***1!C"2(#'!
!
!
PROG (%)
789:;
)&#/!%!)+#/!
)'#"!%!)"#'!
))#*!%!)&!
)(#&!
(#())!
=;89!.!>?
)+#"!.!(#+!
)'#/!.!(#&!
)'#/!.!)#'!
>@:9@A@B89B;
C)2!(#('*1!0'2!#('1!0"2)!
VAP (m/s)!
789:;
D"#,*!%!+#$E)(-6!
D'#"!%!'#,E)(-6!
D"#,!%!&#&E)(-6!
)"#*,!
(#(($!
=;89!.!>?
D&#/!.!(#/E)(-6!
D'#$!.!(#"E)(-6!
D&#)!.!(#'E)(-6!
>@:9@A@B89B;
C)2!#(($**1!0'2!(#+1!0"2(#("*!
VCL (m/s)!
789:;
D*#"!%!))#&E)(-6!
D+#"!%!$#*E)(-6!
D/!%!/#'E)(-6!
+#+!
(#(&!
=;89!.!>?
D,#"!.!'E)(-6!
D+#,/!.!(#*E)(-6!
D/#)!.!(#)E)(-6!!
>@:9@A@B89B;
C)2!(#(+*1!0'2!(#,1!0"2!(#'!
VSL (m/s)!
789:;
D)#+!%!)#,E)(-6!
D(#,!<!)#'E)(-6!
D)#',!%)#&E)(-6!
)&!
(#((+!
=;89!.!>?
D)#*!.!(#'E)(-6!
D)#)!.!(#)E)(-6!
D)#"!.!(#)E)(-6!
>@:9@A@B89B;
C)2!(#(($**1!0'2!(#(/1!0"2!(#'!
STR (%)!
789:;
"&#"!%!"*#*!
"(#"!%!"+#)!
'"#/!%!""#&!
"#,!
(#(/&!
=;89!.!>?
"+#,*!.!)#*!
"'#*!.!'#&!
'/#$!.!&#/!
>@:9@A@B89B;
C)2!(#/1!0'2!(#)1!0"2!(#+!
LIN (%)!
789:;
=;89!.!>?
)+#,!%!'(#*!
)'#*!%!)+#+!
)(#,!%!)$#+!
"#*!
(#(//!
)/#"!.!'#&!
)&#)!.!)#&!
)"#*!.!'#/!
>@:9@A@B89B;
C)2!(#'1!0'2!(#)1!0"2!)!
WOB (%)!
789:;
&$#'!%!+"#*!
")#"!%!"*#"!
"&#'!%!&&#'!
))#,!
(#((/!
=;89!.!>?
+(!.!"#/!
"&#"!.!'#,,!
",#'!.!+!
>@:9@A@B89B;
C)2!(#((,**1!0'2!(#(+*1!0"2!(#+!
ALH (m)!
789:;
D$!%!$#)"E)(-6!
D&#&*!%!+#')E)(-6!
D&#&$!<!+#,$E)(-6!
+#(+!
(#(+'!
=;89!.!>?
D$#)!.!(#($E)(-6!
D&#/!.!(#&E)(-6!
D+#'!.!(#/E)(-6!
>@:9@A@B89B;
C)2!(#($1!0'2!(#'1!0"2!)!
BCF (Hz)!
789:;
'#'+!%!'#+'!
'#($!%!'#&*!
'#(,<'#'/!
)#"!
(#"!
=;89!.!>?
'#&!.!(#)!
'#"!.!(#'!
'#'!.!(#(,!
>@:9@A@B89B;
C)2!)1!0'2!(#&1!0"2!)!
34560!FG!69;H05I;J1!34560!FFG!K5L<J5I;!:4560!89J!34560!FFFG!M@:M<J5I;!:4560#!01G!0!N8K6;!A54!B5O084@9:!P;QL;;9!:4560!F!89J!:4560!
FF1!02G!0!N8K6;!A54!B5O084@9:!P;QL;;9!:4560!F!89J!:4560!FFF1!03G!0!N8K6;!A54!B5O084@9:!P;QL;;9!:4560!FF!89J!:4560!FFF1!RG! >Q8Q@IQ@B8KKS!
I@:9@A@B89Q! 8Q! 0! T! (#(+1! RRG! >Q8Q@IQ@B8KKS! I@:9@A@B89Q! 8Q! 0! T! (#()1! RRRG! >Q8Q@IQ@B8KKS! I@:9@A@B89Q!8Q! 0! T! (#(()#! D=UVG! O5Q@K@QS1! C7U3G!
045:4;II@N;!O5Q@K@QS1!WXCG!N;K5B@QS!8N;48:;!08QM1!WYZG!N;K5B@QS!B64N;J!K@9;1!W>ZG!N;K5B@QS!IQ48@:MQ!K@9;1!>V7G!IQ48@:MQ9;II1!ZF[G!K@9;84@QS1!
\U]G!L5PPK;1!XZ^G!8O0K@Q6J;!5A!K8Q;48K!M;8J!J@I0K8B;O;9Q1!]Y_G!P;8Q!B45II!A4;`6;9BSE#!
Tab le 3. Sperm morphology analysis
Group I (n = 15)
Group II (n = 15)
Group III (n = 15)
F
P
>?F!
789:;!
)#+*!%!'!
"#)*!%!"#$!
'#"!%!"#'+!
),#"$!
(#(('!
!
=;89!.!>?!
)#/!.!(#'!
"#&!.!(#'!
'#*,!.!(#+!
!
!
!
>@:9@aB89B;!
C)2(#(("R1!C'2(#('R1!C"2(#'!
!
!
[54O8K!I0;4OI!DbE!
789:;!
)&!%!)&#"!
*#'!%!))#,!
$#+!%!))#$!
+#/,!
(#("/!
!
=;89!.!>?!
)&#)+!.!(#)!
,#$!.!'#&!
,!.!'#+!
!
!
!
>@:9@aB89B;!
C)2!(#("R1!0'2!(#('R1!0"2(#/!
!
!
V;4 8 Q 5 ! I 0 ;4OI!Db E !
789:;!
/+#*!%!/$!
//!%!,'#/!
//#&!%!,"#'!
+#/,!
(#("/!
!
=;89!.!>?!
/+#/!.!(#)!
,(#&!.!'#&!
,)!.!'#+!
!
!
!
>@:9@aB89B;!
C)2!(#("R1!0'2!(#('R1!0"2(#/!
!
!
34560!FG!69;H05I;J1!34560!FFG!K5L<J5I;!:4560!89J!34560!FFFG!M@:M<J5I;!:4560#!0)G!0!N8K6;!A54!B5O084@9:!P;QL;;9!:4560!F!89J!:4560!
FF1!0'G!0!N8K6;!A54!B5O084@9:!P;QL;;9!:4560!F! 89J!:4560! FFF1!0"G!0!N8K6;!A54!B5O084@9:!P;QL;;9!:4560!FF!89J!:4560!FFF!RG!>Q8cIcB8KKS!
I@:9@aB89Q!8Q!0!T!(#(+#!D>?FGI0;4O!J;A54O@QS!@9J;HE#!
El Sewedy et al., 2023
IJCBR Vol. 7(2): 35-45
40
Figure 3. Bar chart representing the mean value of glucose
levels (mmol/L) in all studied groups. Non-significant
difference was ignored. ***: Statistically significant at p ≤
0.001.
Figure 4. Bar chart representing the mean value of CRP
levels (mg/L) in all studied groups. n=15. **: Statistically
significant at p ≤ 0.01, ***: Statistically significant at p ≤
0.001.
The representative microphotographs of sperm
morphology in all studied groups are shown in
Figure 2.
Effect of IS on blood glucose level
The mean blood glucose levels (mmol/L) are
illustrated in Figure 3. Incense smoke inhalation
caused a significant decrease in blood glucose
levels in low and high-dose IS inhaling groups
compared to the control unexposed group (P<
0.001). The mean blood glucose levels in the low
and high doses and control unexposed groups
were 7.9 ± 0.4, 7.8 ± 0.4, and 9.4 ± 0.6,
respectively. On the other hand, the mean IS
blood glucose levels in the high-dose group
were insignificantly lower than that in the low-
dose group (P= 0.945).
Effect of IS on C-reactive protein (CRP) level
The range and mean ± SD of CRP (mg/L) in the
studied groups (Figure 4). Incense smoke
caused a dose-dependent significant increase in
CRP levels compared to the control group (P<
0.001). The mean CRP levels in low, high dose,
and control groups were 25.6 ± 6, 35.3 ± 4.1,
and 5.5 ± 2.7, respectively.
Effect of IS on kidney function
As presented in Figure 5A, there were
statistically significant differences in the mean
urea levels (mmol/L) between the studied
groups. The mean urea levels in low and high-
dose groups were dose-dependent and were
significantly higher (P< 0.001) than that in the
unexposed group (7.3 ± 0.6, 7.9 ± 0.3, and 5.5 ±
0.1, respectively). Similar to urea levels, the
mean creatinine levels (µmol/L) in low and high-
dose exposed groups were significantly higher
(P< 0.001) compared to the unexposed group
(50.4 ± 2.7, 53.05 ± 2.7, and 29.2 ± 2.7,
respectively) (Figure 5B). However, there was
an insignificant difference between creatinine
levels in low-dose and high-dose groups (P=
0.275).
Effect of IS on liver function
Statistically significant differences in the mean
ALT levels (U/L) were detected between the
three studied groups (P< 0.001). The mean ALT
levels in low and high-dose groups were
significantly higher than that in the unexposed
group (204.9 ± 32.41, 287.6 ± 25.37, and 107.2
± 6.19, respectively) (Figure 6A). Moreover, the
mean ALT level in the high-dose group was
significantly higher than that in the low-dose
group (P< 0.001). On the other hand, and similar
to ALT levels, the means AST levels (U/L) in low
and high dose groups were significantly (P<
0.001) higher than that in the control group
(135.4 ± 2.46, 132.7 ± 5.64 and 114.3 ± 7.02,
respectively). However, the low-dose and high-
dose groups showed statistically insignificant
differences (P= 0.621) (Figure 6B).
DISCUSSION
We showed that serum testosterone levels and
sperm quality were negatively affected by
prolonged IS inhalation in a dose-dependent
manner.
0
2
4
6
8
10
12
Control Low dose High dose
Mean value of glucose(mmol/L)
***
***
0
5
10
15
20
25
30
35
40
45
Control Low dose High dose
Mean value of CRP (mg/L)
***
***
**
Incense smoke inhalation affects spermatogenesis and sperm quality and impairs liver and kidney function in adult male rats
IJCBR Vol. 7(2): 35-45
41
Figure 5. Bar chart representing the mean value of urea
levels (mmol/L) (A) and creatinine levels (µmol/L) (B) in all
studied groups. The non-significant difference was
ignored. *: Statistically significant at p ≤ 0.05, ***:
Statistically significant at p ≤0.001.
Figure 6. Bar chart representing the mean value of ALT (A)
AST (B) levels (U/l) in all studied groups. ***: Statistically
significant at p ≤ 0.001.
Moreover, prolonged inhalation of IS caused
significant potential alterations in liver and
kidney biochemical markers, suggesting
potential toxicity. Regardless of the incense
type, several studies have shown that emissions
of air pollutants from burning incense contain
many deleterious components including
inorganic gases, particulate matter (PM), toxic
organic compounds, and heavy metals (Jetter et
al., 2002; Lee & Wang, 2004; Yang et al., 2012).
Inorganic gas such as CO is known to cause
toxicity due to its high affinity for hemoglobin,
which causes a decrease in oxygen supply to
tissues, resulting in hypoxia (Palmeri & Gupta,
2022).
Environmental exposure to hypoxia was
reported to cause alterations in blood flow and
oxygen supply along with an increased local
temperature that may affect sperm quality
(Reyes et al., 2012). Nitric oxide (NO), released
from different types of incense was as high as
0.3 ppm, which is significantly higher than the
acceptable levels of NO (<0.080 ppm) (Bootdee
& Chantara, 2014). NO was found to induce
mouse infertility in a time-dependent manner.
Oxidative stress and apoptosis are involved in
mediating NO-induced infertility (Wu et al.,
2022). Nitrogen Dioxide (NO2) and Sulfur
dioxide (SO2) induced the expression of
oxidative-stress-related genes and were
negatively associated with sperm quality in a
population of men with infertility (Chen et al.,
2020). Both NO2 and SO2 concentrations
emitted from incense burning were found to
surpass the standards stated by US National
Ambient Air Quality Standards (NAAQS) (Jetter
et al., 2002). Given the slow-burning process of
incense, significant PM of different sizes is
produced, and air pollution containing PM was
previously reported to damage the human-
sperm sex ratio (Radwan et al., 2018). The most
common mechanism of action for all released
toxins on spermatogenesis was systemic
increases in oxidative stress, which in turn leads
to aberrant inflammation and irreversible
sperm DNA impairment, therefore affecting
male fertility (Alahmar, 2019; Zhang et al., 2019;
Huang et al., 2020; Kurkowska et al., 2020).
Maintenance of the structure and function of
the male reproductive system are androgen
dependent. Testosterone is necessary for the
0
1
2
3
4
5
6
7
8
9
Control Low dose High dose
Mean value of urea (mmol/L)
***
*
***
0
10
20
30
40
50
60
Control Low dose High dose
Mean value of creatinine (µmol/L)
***
***
0
50
100
150
200
250
300
350
Control Low dose High dose
Mean value of ALT leves (U/l)
***
0
20
40
60
80
100
120
140
160
Control Low dose High dose
Mean value of AST levels (U/l)
***
El Sewedy et al., 2023
IJCBR Vol. 7(2): 35-45
42
spermatogenesis process and male fertility
(Merghem et al., 2017; Walker, 2021).
Therefore, any abnormalities and changes
observed in this system may be linked to a
testosterone deficiency (Walker, 2021). Our
results indicated that testosterone levels in IS-
exposed rats were significantly lower than that
in the unexposed group. Exposure to air
pollutants such as PM, CO and NO was reported
to be negatively associated with the level of
testosterone in a study performed on 327 men
attending an infertility clinic (Radwan et al.,
2016).
In andrology, it is believed that the main
determinant of male fertility outcome is semen
quality (Bonde et al., 1998). Computer-assisted
semen analysis (CASA) showed that sperm
progressive motility, velocity parameters, and
vigor parameter (wobble %) significantly
declined in IS-exposed rats compared to the
unexposed group. The percentage of
morphologically abnormal sperms significantly
increased compared to the unexposed group.
The sperm deformity index value in low-dose
and high-dose groups was significantly higher
than that in the unexposed group. Based on a
study that demonstrated that fragile DNA
showed a higher SDI value and vice versa (Aziz
et al., 2011), our results indicate a more fragile
DNA in IS-exposed groups. These may be due to
the direct effect on testicular tissue which
results in reproductive dysfunction such as
reduced sperm progressive motility, velocity,
and vigor parameters as well as sperm
morphological abnormalities. A previous report
demonstrated that Carlina gummifera IS
inhalation caused oxidative stress in the testis,
which may be the indirect cause of the observed
changes in spermatogenesis (Dorsaf et al.,
2022).
Blood glucose is an indicator of the healthy state
of the organism, our results indicated that blood
glucose level was significantly decreased in IS-
exposed rats compared to the unexposed
group, suggesting liver damage as recently
indicated by Bai et al. study which
demonstrated that the levels of fasting blood
glucose and 2 h postprandial glucose changed in
liver cirrhosis. Glycogen concentrations and the
activity of glucose -6-phosphatase in the liver
were decreased (Bai et al., 2021).
Epidemiological studies suggest that exposure
to carbon monoxide, nitrogen dioxide and PM
from outdoor or traffic-related air pollution is
related to a greater risk of chronic kidney and
liver disease (Kim et al., 2019). The kidneys and
liver's normal function is not only important for
physiological processes such as detoxification,
metabolism, protein synthesis, blood sugar
balance, waste and excess fluid elimination and
regulation of blood pressure but most
importantly to the scope of this manuscript,
kidney or liver disease were reported to affect
sperm production and quality (de Kretser,
1997). Serum hepatic enzyme activity was
negatively related to sperm concentration and
total sperm count (Ehala-Aleksejev & Punab,
2016). Moreover, sexual and reproductive
function was reported to be remarkably
affected in chronic kidney disease (Mahboob &
Shirin, 2011; Dumanski & Ahmed, 2019).
Regarding the effect of IS inhalation on liver
function, results indicated that blood ALT and
AST levels were significantly increased in IS-
exposed rats compared to the unexposed
group. This suggests that IS inhalation may
cause liver damage where the liver is
susceptible to damage from direct exposure to
toxic products due to excessive detoxification of
toxins and xenobiotics (Abd-Elhady & Abou-
Elghar, 2013). It was demonstrated that wood
smoke PM can cause liver damage through the
generation of reactive oxygen species, lipid
peroxidation, genotoxicity and endoplasmic
reticulum stress (Danielsen et al., 2010; Laing et
al., 2010).
Urea and creatinine are excreted primarily by
the kidney so their blood levels serve as
indicators of renal function. Blood urea and
creatinine levels were significantly increased in
IS-exposed rats compared to the unexposed
group in a dose-dependent manner. This finding
suggests that IS inhalation may be associated
with a disturbance of renal function. The
mechanism underlying the link between IS
inhalation and renal disease might be explained
by Hussain et al study, which indicated that IS-
exposed rats had deterioration in normal
physiological functions with a substantial
increase in serum creatinine, uric acid, and
blood urea nitrogen, and significant ultra-
structural changes in kidney tissues. This was
Incense smoke inhalation affects spermatogenesis and sperm quality and impairs liver and kidney function in adult male rats
IJCBR Vol. 7(2): 35-45
43
accompanied by a corresponding increase in
inflammatory markers such as tumor necrosis
factor-alpha and interleukin-4 levels, an
increase in oxidative stress indicated by a
significant increase in tissue malondialdehyde
and tissue gene expression of both CYP1A1 and
CYP1A2, and a decline in tissue reduced
glutathione and catalase activity (Hussain et al.,
2016).
Additionally, our study showed that the mean of
serum CRP levels in IS-exposed rats was
significantly higher than that in the unexposed
group in a dose-dependent manner. Moreover,
it was reported that CRP is not only an
inflammation biomarker but also an activated
mediator of acute kidney injury. It exerts
undesirable effects on the injury processes and
affects repairing through multiple mechanisms
(Tang et al., 2018). Collectively, we suggest that
the adverse modulating effects of IS inhalation
on renal functions in rats may be mediated
through augmented inflammation and oxidative
stress.
Collectively, our findings indicated that IS
inhalation may cause kidney and liver damage.
Moreover, it can affect sperm quality including
progressive motility, velocity, and vigor
parameters, and cause abnormal morphological
changes. However, further studies are required
to explain the molecular mechanism of IS-
induced cytotoxic effects.
CONCLUSION
This study shows that prolonged IS inhalation
may affect male fertility potential where it
significantly reduced sperm progressive
motility, velocity, and vigor parameters and also
significantly affect sperm morphology.
Moreover, it significantly affects kidney and
liver function as well as blood glucose levels.
ETHICS APPROVAL AND CONSENT TO
PARTICIPATE
All animal procedures and experimental
protocols were approved by the Research Ethics
Committee of the medical research institute,
Alexandria University (AU0122282233). All
animal experiments were performed according
to the ARRIVE guidelines and the National
Research Council’s guide for the care and use of
laboratory animals.
AVAILABILITY OF DATA AND MATERIALS
The datasets used and/or analyzed during the
current study are available from the
corresponding author upon reasonable request.
COMPETING INTERESTS
The authors declare that they have no
competing interests.
FUNDING
The study is self-funded.
AUTHORS' CONTRIBUTIONS
T.S. & N.F. designed the research proposal idea
and performed the practical part of the
research. N.F. and S.E. analyzed and interpreted
the results of this study. T.S. and S.E. wrote the
main manuscript text. All authors read and
approved the final manuscript.
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