Comparative Clinical Pathology

https://doi.org/10.1007/s00580-023-03513-x

ORIGINAL ARTICLE

Cement dust inhalation induces hepato‑renal dysfunction via tissue heavy

metal bioaccumulation, histopathological and biochemical mechanisms
M. W. Owonikoko1   · A. T. Salami1 · A. O. Odukanmi1 · B. O. Emikpe2 · S. B. Olaleye1

Received: 28 July 2022 / Accepted: 14 August 2023

© The Author(s), under exclusive licence to Springer-Verlag London Ltd., part of Springer Nature 2023

Abstract
Systemic degradations of various forms have been adduced to cement dust exposure and Inhalation is the most
considerable route. We deployed inhalation method to investigate the possible reno-hepatic effect of cement dust
exposure. Thirty male Wistar rats weighing between 150 and 180g acquired were acclimatized and randomized into
three groups of 10 animals each. Group 1 was the control, group 2: 14-day exposed, and group 3: 28-day exposed.
Relative organs weights, heavy metal analysis, hepatic and renal functions tests, fibrotic index, lipid peroxidation
(MDA), superoxide dismutase (SOD), catalase (CAT), nitric oxide (NO), sulfhydryl group, histopathology, and
histomorphometry were assessed using standard procedures. There was increase in organ weight and significant
(p < 0.05) tissue bioaccumulation of heavy metals. There were significant increases in urea, creatinine, hepatic aspar-
tate aminotransferase, Alanine aminotransferase and Alkaline phosphatase values in the exposed groups compared
with control. Inhaled cement dust was observed to induce lipid peroxidation and deplete SOD and CAT levels. There
was also significant upregulation of renal and hepatic NO in the 14-day (0.38 ± 0.05 nmol/g to 0.55 ± 0.17 nmol/g)
and 28-day (0.48 ± 0.11 nmol/g to 0.80 ± 0.04 nmol/g) groups when compared with their respective control groups
(0.78 ± 0.01 nmol/g to 1.33 ± 0.17 nmol/g). A significant reduction in renal and hepatic sulfhydryl groups was also
observed in the exposed groups when compared with the controls. Histopathology showed an array of alterations
ranging from vascular congestion, necrosis to neutrophil infiltration. Cement dust exposure by inhalation induced
reno-hepatic functional alteration via increased organ weight, heavy metal bioaccumulation, histopathological, and
biochemical mechanisms. Biochemically, cement dust inhalation depleted antioxidant capacity and generated oxida-
tive and nitrosative stress.

Keywords  Cement dust exposure · Liver toxicity · Kidney toxicity · Oxidative stress · Antioxidant status

Introduction (Laniyan and Adewumi 2020; Olatunbosun et  al. 2020).

Cement dust is a depository of toxicants including toxic met-
als and metalloids. Cement factory workers are exposed while
the safety of people living within the territories of cement fac-
tories is equally not guaranteed as their environment is known
to be contaminated with a very high level of heavy metals
* M. W. Owonikoko
owonikoko.mathew@yahoo.com
* S. B. Olaleye
sbolaleye@yahoo.com
1
Department of Physiology, College of Medicine, University
of Ibadan, Ibadan, Nigeria
2
Department of Veterinary Pathology, Faculty of Veterinary
Medicine, University of Ibadan, Ibadan, Nigeria
Recently, Ugbaja and his colleagues reported that snails
thriving in cement factory vicinities bioaccumulate toxic
elements beyond the permissible limit (Ugbaja et al. 2020).
Investigation on cement dust-induced pathologies is required
in this modern period plagued with increased environmental,
occupational, and geographical exposure characterized by
a significant atmospheric abundance of heavy metals. The
toxicodynamics and the physicochemical interaction of toxi-
cants with the body tissues can be significantly altered par-
ticularly when toxicants are co-administered (Brzóska et al.
2003; Ezedom et al. 2020). Hence, heavy metal co-exposure
mimics the reality of human exposure than individual heavy
metal exposure thereby making it a salient issue in contem-
porary toxicology.

13
Vol.:(0123456789)

Cement factories, distribution processes, and host com-
munity vicinities are the primary sources of exposure to
cement dust which happens predominantly via the dermal
route. Since cement dust is usually aerosolized, inhalation is
another significant route of human exposure. The specific-
ity of inhaled materials on the respiratory systems is due to
its direct connection to the external environment; a condi-
tion causing large dust particles, on the one hand, to settle
at the upper respiratory tract while mucociliary escalator
(composed of phagocytes) evacuate them into the gastro-
intestinal tract (GIT) by coughing or swallowing. Such
materials eventually escape into the blood during intestinal
micro-circulation. Fine dust particles, on the other hand,
settle deep down the respiratory tract and as such escape
the phagocytic action of alveolar macrophages. Although
the extrapulmonary toxicokinetics of inhaled xenobiotics
is not clear, their transmigration is well documented (Lee
et al. 1985; Masoudi et al. 2020). It is now putative that
the pulmonary deposition and retention of fine particles is
accompanied by percolation of the alveolar wall and lung-
associated lymph nodes (Ferin et al. 1992); the fate of which
is systemic redistribution where they can access various
other organs via blood supply. This is particularly accurate
for cement dust particles whose heavy metallic constituents
are known to bioaccumulate in tissues following exposure
(Cooper et al. 2017; Ugbaja et al. 2020; Owonikoko et al.
2021). Cement dust has been severally reported to be a
systemic toxicant inducing multi-organ injuries (Richard
et al. 2016), bone-marrow mediated hematopoietic toxicity
(Owonikoko et  al.  2021, 2022), pulmonary pathological
changes, and heavy metal histo-bioaccumulation (Owonikoko
et al. 2021). However, there had hitherto been a dearth of lit-
erature information on the effect of cement dust on hepatic
and renal functions, particularly in experimental conditions.
The kidney and liver are the two most perfused visceral
organs in the body with an approximate supply of 25% and
28% cardiac output respectively from the arterial tray (Barrett
et al. 2019). Owing to their salient roles in general body
metabolism and homeostasis, these humungous perfusion rates
are inevitable and therefore potentiate their susceptibility to
xenobiotic content of the blood including heavy metals. This
is because exogenous toxicants are translocated via the instru-
mentation of the hematological system prior sequestration
and bioaccumulation—processes known to determine the fate
of internalized toxicants. Liver and kidney share similarities
in a number of physiological processes including secretory,
endocrine, and hematopoietic functions. They also prevent
deleterious systemic fluctuations by regulating, modulating,
and maintaining internal milieu. These apparent physiological
provisions overtly predispose the organs  to xenobiotic-
induced impairments than other visceral organs (Nair
et al. 2016; Abdel-Daim and Abdeen 2018). They are usually
the specific targets in free radicals-induced organ injuries.
13
Comparative Clinical Pathology
Till present, there is dearth of in vivo studies (characterized
with the use of a controlled experimental exposure method)
investigating systemic cement dust toxicity particularly with
respect to liver and kidney in spite of the plethora of evidence
about cement dust toxicity. It is unclear whether the liver and
the kidney are susceptible to dysfunctions when exposed to
cement dust by the inhalation route. Therefore, estimating the
hazard potential of cement dust in order to facilitate safety
decisions, its effect on the liver and kidney will be a reliable
systemic toxicity index. This study therefore investigates reno-
hepatic dysfunctions due to cement dust exposure by inhalation.
Materials and method
Cement material and experimental exposure
Full, intact and freshly supplied bag of Nigeria Portland
cement was acquired from an accredited company depot
within the neighborhood of the University of Ibadan,
Ibadan, Nigeria. The animals were experimentally exposed
to cement dust using the method described by Nwafor et al.
2019 and standardized by Owonikoko et al. 2021 using a
plexi-glass house chamber for dust exposure to plantigrades
was used. The chamber consists of two (2) distinct, effec-
tively partitioned but complementary systems viz: the ani-
mal housing system and the dust management system. The
latter consist of sub-divisions including the ‘dust housing
system’ and the ‘aeration system’. While the dust housing
system accommodates only the dust of known quantity and
have been effectively partitioned to prevent the entry of the
experimental animals, animal housing system accommo-
dates only the experimental animals and the aerations system
consist of two metallic aerators (serial number S40141392;
model CNS-3-20/620). The latter is capable of generating
and distributing cement dust from the dust housing system
only when connected to the mains, at 220V. The chamber
dispenses cement dust at the effusion rate of 0.2 g/h; the
experimental animals were restricted to the larger com-
partment to prevent extra-inhalational exposure. Exposure
kicked off daily by the deposition of 100 g of cement parti-
cles daily into the sub-chamber. Left over cement particles
from previous day’s exposure were completely and consist-
ently ridded of the chamber.
Chemicals
All reagents such as Nitric acid, ethanol, methanol and phos-
phate buffer preparation salts used were of analytical grade
and were obtained from British Drug Houses, Poole, UK
and Mumbai, India
Comparative Clinical Pathology
Experimental animals, groupings, and study approval
Thirty (30) mature male Wistar rats weighing between 150
and 180 g were acquired from the central animal house of
the college of medicine, Faculty of Basic Medical Science,
University of Ibadan. The experimental animals were accli-
matized for the period of 2 weeks following arrival and ran-
domized into three (3) groups of 10 animals each accord-
ing to the level of cement dust exposure. Group 1 animals
were raised in an environment completely free of dust; they
served as control, having received sham exposure to dust-
free atmospheric oxygen. Group 2 animals were exposed to
cement dust for a total of 14 days (14-day exposed) while
group 3 were exposed to cement dust for 28 days (28-day
exposed). Each exposure was carried out 5 h daily between
8 am and 1 pm. All procedures used are in line with the
Animal Care Regulations and Standards as approved by the
Institute for Laboratory Animal Research (Hermenean et al.
1996) while the experimental protocols were approved by
the Animal Care and Use Research Ethics Committee of
the Faculty of Veterinary Medicine, University of Ibadan,
Ibadan and allotted and documented with the approval num-
ber UI-ACUREC/18/0129.
Body weight, organs harvest, and organ weights
At sacrifice, the instantaneous body weight of each rat was
taken while liver and kidney organs weights were also taken
after harvesting with the aid of ­Acculab® USA, Model-
vic-303 electronic analytical weighing balance. The per-
centage relative organ weight (%ROW) was calculated by
using the formula;
%ROW = {(X∕Y) × 100}g (1)
where ‘X’ represents the ‘Absolute Organ Weight’; the raw
weight of the organ as obtained from the weighing balance
while ‘Y’ is the ‘Terminal Body Weight’; the instantaneous
weight of the animal at the point of sacrifice.
Venesection
On the last day of exposure, after body weight determination,
2.5 ml of blood was carefully drained via orbital venous sinus
bleeding according to the method of Parasuraman et al. 2010,
left to stand in order to clot and the serum was decanted off for
the estimation of liver and kidney function variables.
Digestion of tissues and heavy metal analysis
Renal and hepatic heavy metal levels were investigated
according to Akram et al. 2015. One milliliter each of nitric
acid and perchloric acid were added to 100 mg of the tissues
each in clean sample bottles. The mixture was digested over
a sand bath until the solution turned clear and yellow. The
digests were aliquoted after being made up to known volume
with ionized water. Absorbance was read using Buck Scien-
tific Atomic Absorption spectrophotometer model 210/211
VGP, at various wavelengths according to the standard work-
ing parameters stated in Table 1 below. Results of accumu-
lated heavy metals were recorded in mg/L and presented as
mean ± SEM. Radiation sources were the hollow cathode
lamp of Pb, Cr, Cd, Ni, Fe, Mn, Co, and Ca while the fuel
was air acetylene.
Hepatic and renal response analysis
Hepatic and renal biochemistry
Reno-hepatic functions such as total protein, bilirubin,
urea, creatinine, alanine aminotransferase (ALT), alkaline
phosphatase (ALP), aspartate aminotransferase (AST),
cholesterol, albumin were assessed from the serum using
the RANDOX kits with the respective catalogue numbers
TP 245, BR 411, UR 1068, CR 510, AL 100, AP 542, AS
101, CH 200, and AB 362 having followed manufacturer’s
instructions stringently. ALT and AST were determined
based on colorimetric principle using the respective above
stated commercially available kits. Serum total protein was
determined according to the method of Lowry et al. 1951.
Concentrations of bilirubin, urea, creatinine and activity of
ALP were evaluated using diagnostic laboratory protocol
and determined spectrophotometric method.
Table 1  Specifications for atomic absorption spectrophotometer’s
operation during heavy metal analysis
S/No Metal Wavelength (nm) Slit width
1 Cadmium 228.9 0.7
2 Chromium 357.9 0.7
3 Lead 283.3 0.7
4 Cobalt 352.7 0.7
5 Manganese 279.5 0.7
6 Iron 248.3 0.7
13

Apri Index
This index was evaluated according to El-Wakeel et al. 2018.
Briefly, it is an index derived from the standard formular—
the ratio between AST and platelet count. The result is pre-
sented in percentages.
Homogenate preparation
The organs (liver and kidney) of a total of 5 animals from
each group were carefully excised and rinsed, following sac-
rifice, in normal saline under ice condition and homogenized
(Power plus; Model: PP4113SUL) with phosphate buffer in
the ratio 1:10 (w:v) and cryo-centrifuged at – 10 °C (CRU-
5000 Centrifuge). The supernatant was thereafter decanted
and used for analysis.
Oxidative stress assessment
Tissue total protein
Total protein concentrations of the liver and kidney were
estimated according to the method of Lowry et al. 1951. 3ml
Biuret reagent was aliquoted into 1 ml of the supernatant
from the sample homogenate. The mixture was thereafter
incubated (at room temperature) for 30 min. The absorbance
was read thereafter at 540 nm with the aid of a microplate
reader with bovine serum albumin as the standard.
Lipid peroxidation
This study assessed the extent of lipid peroxidation/MDA
level in the liver and kidney homogenates (0.4 mls each)
by measuring the thiobarbituric acid reactive substances
(TBARS) according to the method of Varshney and Kale
(1990). At 100 °C and in acidic medium, TBARS reacts
with MDA precipitating a red/pink color change which is
extracted by butanol. Absorbance of the clear supernatant
was read at 532 nm using a microplate reader (Epoch BioTek
Instruments, Inc.)
Superoxide dismutase (SOD) activity
Intracellularly, asides the in vivo biodegradational ability of
oxygen in a process of making energy available from the body
system from chemical fuel, it is considered a dangerous chemi-
cal species to most cellular processes in the body as it also
able to generate superoxide radicals by binding and ripping of
13
Comparative Clinical Pathology
electron from them. SOD is an endogenous antioxidant enzyme
which scavenges for the superoxide radicals. Total SOD activ-
ity was assessed in the homogenate samples in accordance
with the indirect method described by Oyanagui 1984. Xan-
thine oxidase enzyme was used to catalyzes the production of
superoxide anion. The latter was reacts with hydroxylamine
producing nitroso ion which combines with sulfanilic acid and
n-(1-naphthyl) ethylenediamine. The reaction which precipi-
tated a characteristic color measured spectrophotometrically
at the wavelength of 560nm and recorded. A unit of SOD is
taken as the amount of the SOD enzymes required to inhibit
tetrazolium reduction
Catalase
The level of catalase was assayed according to the method of
Goth 1992. Perhydrol in 50 mM TRIS/HCl buffer was added
to the homogenized and buffered liver or kidney samples
(0.25 ml each) and the pH adjusted to 7.4. The reaction was
primed by the addition of fresh hydrogen peroxide (­ H2O2;
BDH). Since water and Oxygen do not absorb at 240 nm
as ­H2O2 does, the rate of H­ 2O2 decomposition was spec-
trophotometrically read at that wavelength using UV Vis
Spectrophotometer (Model: UV1700).
Inflammatory and cytomembrane integrity
Nitric oxide (NO)
Greiss reaction (Sigma, Poole, UK) and the level of nitric oxide
were assessed based on the level of its metabolites (Nitrite
and nitrate) in the tissue supernatant according to Karampour
et al. 2019. Renal and hepatic homogenates samples (0.6 ml
each) were each diluted in phosphate buffered saline—an
isotonic buffer comprising of NaCl (CAS:17647-14-5 LOT
NO: 20130713); KCl (Kermel UN Number:1690), anhydrous
Sodium phosphate dibasic (CAS:7558-79-4 LOBA Chemie
PVT Ltd.) and Potassium phosphate anhydrous (Molychem,
Mumbai from India batch no.: MCR- 18108 case no.: 7758-
11-4). Nitrate content was converted into nitrite with the aid of
nitrate reductase- a process followed by the addition of Greiss
reaction 1 [sulfanilamide (1%)] in conc. phosphoric acid (5%)
(BDH) which ultimately yields azo product with a color change
in the presence of 0.1% N-(1-Naphthyl) ethylenediamine (Gre-
iss reaction 2) in a photo-proof container. The endpoint absorb-
ance was read at 520 nm and values were recorded.
Sulfhydryl
Total thiol content of the hepatic and renal homogenate
samples was evaluated. The sample was homogenized
Comparative Clinical Pathology
and diluted with phosphate buffer by a factor of 10 of
the organ weight each. 0.5 ml of each of the liver and
kidney organ was then aliquoted for sulfhydryl assay.
The method used was as described by Ellman 1959.
Aliphatic thiol compounds are known to react with
Bis(p-nitrophenyl) disulfide (I) at pH 8.0 to produce
one mole of p-nitrothiophenol anion per mole thiol. 95%
5-5-Dithiobis-(2-nitrobenzoic acid) (DTNB; Central
drug House (P) LTD batch no: 040718) also known as
the Ellman’s reagent was prepared by dissolving it in 1ml
ethanol (95%) in the presence of ­N2-deficient Tris buffer
and de-ionized water. The reaction yielded 5-mercapto-
2-nitrobenzoate (MNB) and was aliquoted for reading.
The absorbance of the resulting anion was read using the
microplate at the wavelength of 412 nm.
Histopathology and histomorphometry
The excised organs (kidney and liver) having been care-
fully examined for any gross pathology were fixed in an
adequate solution of formosaline and immediately pro-
cessed for the assessment of possible cytoarchitectural
changes. They were thereafter embedded in paraffin wax;
sectioned at 5 μm and stained with hematoxylin and eosin
before viewing under the light microscope for any patho-
logical alterations according to Haber and Lopez 1999
with the aid of the following scale:
0: morphological change absent in animals in a group; 1:
morphological change rarely found in animals in a group;
Fig. 1  a Effect of cement dust on relative kidney weight. **Significant when compared with the control (F value = 34.72; p value < 0.0001). b
Effect of cement dust on relative liver weight. **Significant when compared with the control (F value = 46.73; p value < 0.0001)
2: morphological change found in few animals in a group;
3: morphological change common to most animals in the
group; 4: morphological change common in all animals in
the group.
Percentage histomorphometric increase was calculated
using the formula given below
VE − VC
× 100
VC
Percentage histomorphometric changes was presented as
the addition of the percentage increase and 100%.
Statistical analysis
Statistical analysis was performed using GraphPad
prism 8.0® software for windows with data presented as
mean ± SEM for n = 8 per group. One-way ANOVA was
used for group comparison with Dunnett’s post hoc test; dif-
ferent levels of significance were stated in each case. How-
ever, p < 0.05 was considered significant.
Results
Figures 1a, b shows the effect of cement dust on the relative
liver and renal weights. The weight of the kidney and liver
significantly increased in the 14- and 28-day groups when
compared with the control. Similar trend was observed for
the kidney.
13
 Comparative Clinical Pathology

Table 2  Effect of cement dust exposure on the level of some serum 0.4

parameters

Total Bilirubin (mg/dl)

*
Serum Control 14-day 28-day P value
parameters 0.3 *
T. protein 7.12 ± 0.16 7.58 ± 0.25 7.28 ± 0.21 0.32
(g/dl) 0.2
Albumin 2.75 ± 0.06 3.18 ± 0.12* 3.18 ± 0.09* 0.02
(g/dl)
Globulin 4.32 ± 0.19 4.40 ± 0.17 4.18 ± 0.12 0.64 0.1
(g/dl)
BUN (mg/ 16.00 ± 0.25 17.08 ± 0.18* 17.00 ± 0.29* 0.02
dl) 0.0
Creatinine 0.67 ± 0.03 0.86 ± 0.07* 1.03 ± 0.03* 0.01 CONTROL 14 DAYS 28 DAYS
Chol (mg/ 49.40 ± 2.84 44.20 ± 1.07 47.60 ± 3.31 0.38 Total Bilirubin
dl)

*Significantly different when compared with the control group Fig. 2  Effect of exposure to cement dust on total bilirubin. *Signifi-
cant when compared to the control (F value = 18.30; P = 0.0010)

Analyses of liver and kidney function tests are shown in

Tables 2 and 3. Figures 2 and 3 show the effect of cement 15
dust exposure on toxicological indices; AST-platelet ratio
(APRI index) and blood urea nitrogen/creatinine (BUN/Cr) *
ratio. Total bilirubin is significantly higher than the control at
APRI 10-4(µl)

both 14-day and 28-day groups (Fig. 2). Result showed that 10

there is a significant increase in APRI index in the exposed
groups when compared with the control (Fig. 3). As depicted *
in Table 2, there is no significant difference between the level
of Total Protein in the exposed groups when compared with 5
the control. Similar trend was observed in the globulin level
and cholesterol levels while in Table 3, alkaline phosphatase
(ALP), alanine transaminase (ALT), and aspartate transaminase
(AST) levels in the exposed groups were significantly higher 0
than their respective control values. Blood urea nitrogen (BUN) CONTROL 14 DAYS 28 DAYS
and creatinine levels were significantly higher in the exposed
APRI INDEX
groups compared with the control. However, BUN-creatinine
ratio (BUN/Cr) was significantly lower in the exposed
groups when compared with the control (Fig. 4). Fig. 3  The fibrotic effect of exposure to cement dust on the liver.
*Significant when compared to the control (F value  =  1.837;
Figure 5a, b shows the effect of cement dust exposure on P = 0.0001)
the hepatic and renal levels respectively of malondialdehyde
(MDA) as an index of lipid peroxidation. The MDA hepatic
and renal levels in the exposed groups significantly increased with control. Figure 7a, b illustrates the renal and hepatic
when compared with their controls. Figure 6a, b shows the catalase (CAT) levels following exposure to cement dust
effect on renal and hepatic superoxide dismutase (SOD) respectively. In the kidney tissue, the level of the enzyme
respectively. The results show that the exposed groups have significantly reduced in the 14- and 28-day groups when
lower levels of the renal and hepatic enzyme when compared compared with the control while in hepatic tissue, the value

Table 3  Effect of cement dust Liver parameters Control 14-day 28-day p value

exposure on the level of liver
enzymes AST (µl) 35.33 ± 0.33 39.33 ± 0.67* 41.75 ± 0.62* 0.00
ALT (µl) 27.33 ± 0.33 32.67 ± 0.88* 33.33 ± 0.88* 0.00
ALP (µl) 110.70 ± 2.72 124.00 ± 1.41* 122.00 ± 2.00* 0.00

*Significantly different when compared with the control group

13
Comparative Clinical Pathology

BUN/CREATININE (mg/dl) 25 and Ni levels showed significant increase at 14- and 28-day
exposed groups while Pb is significantly increased only at
28-day group when compared with the control. There was no
20 * significant difference in the 14-day group of Cd when com-
* pared with the control (Table 5). The Cr, Pb, Co, and Fe levels
15 of the kidney tissue was significantly higher both at 14- and
28-day exposed groups. Others including Cd, Mn are signifi-
cantly higher only at 28-day exposed while there was no sig-
10 nificant difference in the 14-day group of Cd and Mn when
compared with the control. Ni was only significantly higher at
5 14-day exposed group when compared to the control but show
reduction at the terminal 14 days (Table 5).
The respective percentage changes in serum, hepatic
0 and renal biochemical parameters across the groups are
CONTROL 14 DAYS 28DAYS represented in Tables 6 and 7 respectively. There was per-
centage increase in the values of hepatic parameters such
BUN/CREATININE as Total protein, total bilirubin, albumin-globulin ratio,
MDA, SOD, and CAT within day 1 and day 14 of cement
Fig. 4  Effect of exposure to cement dust on BUN/Cr ratio. *Significant dust exposure. However, the additional terminal 14 days
when compared to the control. F value = 9.323, p value = 0.0064)
witnessed a decline in the percentage change asides CAT
and NO that show increase (Table 6). Interestingly, renal
of the enzyme is only significantly lower in the 28-day group parameters show contrasting trend. Table  7 shows that
and is not statistically different in the 14-day exposed group. there was percentage increase in the values of renal param-
Renal and hepatic levels of Total Thiol are shown eters such as MDA, SOD, CAT, and NO within day 1 and
in Fig. 8a, b. The results show that there is significant day 14 of cement dust exposure. The additional termi-
decrease in the total thiol level in renal and hepatic tis- nal 14 days witnessed a progressive percentage increase
sues of the exposed groups when compared with their con- in these parameters. The only exception was sulfhydryl,
trols. The hepatic and renal levels of nitric oxide (NO) in which showed decrease in the terminal 14 days (Table 7).
response to cement dust exposure are illustrated in Fig. 9a, Figure 10A–C shows the photomicrographs of the his-
b respectively. Figure 9a shows that the hepatic level of topathological response in liver tissue while Figure 11A–C
NO significantly increased in the exposed groups when shows the photomicrographs of the histopathological
compared with the control while Fig. 9b shows that the response in renal tissue following exposure to cement dust.
renal level of NO increased only at the exposed groups. The comparative analyses between the exposed groups and
Tables 4 and 5 show hepatic and renal levels of heavy metal the control in each of the organs have been depicted in
following exposure to cement dust. The liver Cr, Co Mn, Fe, Table 8

Fig. 5  a Effect of exposure a b

to cement dust on hepatic
lipid peroxidation. *Sig-
nificant when compared to
15 30
the control (F value = 5.356, *
MDA nmol/mg (106)

p value = 0.0238). b Effect
MDA nmol/mg (106)

of exposure to cement dust

on renal lipid peroxidation. 10 * 20
*Significant when compared to *
the control (F value = 68.84, p
value = 0.0001)
5 *
10

0 0
CONTROL 14 DAYS 28 DAYS CONTROL 14 DAYS 28 DAYS
MDA LIVER MDA KIDNEY

13
 Comparative Clinical Pathology

Fig. 6  a Effect of exposure to a b

cement dust on renal level of 20
superoxide dismutase enzyme. 20

superoxide dismutase
*Significant when compared to

SOD (nm/mg protein)

(nm/mg protein)
the control (F value = 7.035, 15 *
p value = 0.0108). b Effect of 15
exposure to cement dust on * * *
hepatic level of superoxide 10
dismutase enzyme. *Sig-
10
nificant when compared to the
control (F value = 4.758, p 5 5
value = 0.0251)

0 0
CONTROL 14 DAYS 28 DAYS CONTROL 14 DAYS 28 DAYS
SOD KIDNEY SOD LIVER

a b
20000
CATALASE (nm/mg protein)

20000

15000 CATALASE (nm/mg protein) 15000

* *
*
10000 10000

5000 5000

0 0
CONTROL 14 DAYS 28 DAYS CONTROL 14 DAYS 28 DAYS
CAT KIDNEY CAT LIVER

Fig. 7  a Effect of exposure to cement dust on the level of renal on the level of hepatic catalase enzyme. *Significant when compared
catalase enzyme. *Significant when compared to the control (F to the control (F value = 17.57, p value = 0.0019)
value = 18.65, p value = 0.0027). b Effect of exposure to cement dust

Fig. 8  a Effect of exposure to a b

cement dust on renal total Thiol.
SULFHYDRYL (μm/g wet tissue)

*Significant when compared to

SULFHYDRYL ( μm/g wet tissue)

the control (F value = 9.552, 1.0

2.0
p value = 0.0137). b Effect of
exposure to cement dust on 0.8
hepatic total Thiol. *Sig-
nificant when compared to the 1.5
control (F value = 7.927, p 0.6 *
value = 0.0126)
* 1.0
0.4 *
*
0.2 0.5

0.0
CONTROL 14 DAYS 28 DAYS 0.0
CONTROL 14 DAYS 28 DAYS
SULF KIDNEY
SULF LIVER

13
Comparative Clinical Pathology

a
150 b
600
*
*
NITRIC OXIDE ( μM )

NITRIC OXIDE ( μM )
100 * 400
*

50 200

0 0
CONTROL 14 DAYS 28 DAYS CONTROL 14 DAYS 28 DAYS
NO LIVER NO KIDNEY

Fig. 9  a Effect of exposure to cement dust on hepatic nitric oxide nitric oxide level. *Significant when compared to the control (F
level. *Significant when compared to the control (F value  =  11.50, value = 22.55, p value = 0.0001)
p value  =  0.0033). b Effect of exposure to cement dust on renal

Table 4  Heavy metal analysis of liver tissue (mg/L) of experimental animals exposed to cement dust
Group Pb Cr Cd Co Mn Fe Ni

Control 0.74 ± 0.10 0.03 ± 0.01 0.03 ± 0.02 0.01 ± 0.00 0.03 ± 0.00 0.40 ± 0.10 0.02 ± 0.00

14 days 0.85 ± 0.05 0.38 ± 0.08* 0.05 ± 0.02 0.28 ± 0.07* 0.30 ± 0.10* 1.48 ± 0.02* 0.11 ± 0.02*
28 days 1.31 ± 0.19* 0.93 ± 0.06* 0.35 ± 0.16* 0.77 ± 0.11* 0.56 ± 0.10* 2.07 ± 0.12* 0.40 ± 0.10*
*Significantly different when compared with the control group

Table 5  Heavy metal analysis of kidney tissue (mg/L) of experimental animals exposed to cement dust

Group Pb Cr Cd Co Mn Fe Ni

Control 0.74 ± 0.09 0.03 ± 0.01 0.01 ± 0.00 0.01 ± 0.00 0.03 ± 0.00 0.17 ± 0.08 0.02 ± 0.00

14 days 1.02 ± 0.01* 0.14 ± 0.02* 0.03 ± 0.00 0.31 ± 0.01* 0.07 ± 0.00 1.59 ± 0.16* 0.16 ± 0.03*
28 days 1.32 ± 0.22* 0.21 ± 0.01* 0.47 ± 0.03* 0.43 ± 0.08* 0.29 ± 0.03* 2.83 ± 0.44* 0.08 ± 0.03

*Significantly different when compared with the control group

Table 6  Percentage changes in liver parameters across the group fol-

lowing exposure to cement dust
Parameters Control (%) 14-day (%) 28-day (%) Table 7  Percentage change in kidney parameters across the group fol-
lowing exposure to cement dust
Total protein 100 106 102
Parameters Control (%) 14-day (%) 28-day (%)
Albumin 100 116 116
Globulin 100 102 106 MDA 100 145 179
A:G RATIO 100 117 116 SOD 100 123 137
T. BIL 100 139 12 CAT​ 100 120 135
MDA 100 147 145 NO 100 160 229
SOD 100 128 127 SULF 100 151 139
CAT​ 100 114 123
NO 100 328 388
SULF 100 159 140

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 Comparative Clinical Pathology

13
 Comparative Clinical Pathology
◂Fig. 10  A–C Photomicrographs of liver tissue after inhalatory expo-
sure to cement dust. arrows indicate vascular congestion (B1 and B2),
inflammatory cell infiltration (B1 and B2) and hemorrhagic lesion
(A1 and A2). A1 Liver tissue photomicrograph of rats exposed to
cement dust for 28  days (×  100). A2 Liver tissue photomicrograph
of rats exposed to cement dust for 28  days (×  400). B1 Liver tissue
photomicrograph of rats exposed to cement dust for 14 days (× 100).
B2 Liver tissue photomicrograph of rats exposed to cement dust for
14  days (×  400). C1 Liver tissue photomicrograph of control rats
(× 100). C2 Liver tissue photomicrograph of control rats (× 400)
Discussion
This study is a progressive investigation of the pathophysi-
ological mechanisms involved in cement dust toxicity. We
retained the referenced model of inhalation exposure and
deployed its instrumentation to investigate the possible
extra-respiratory effect of exposure to cement dust with
special interest on liver and kidney.
A total of 30 male Wistar rats were exposed to cement dust
using the chamber for a total of 5 h per day for a total of 14
and 28 days. The days of exposure for this study informs the
grouping system generating a total of 3 groups. Renal and
hepatic tissue weights, functional parameters, and relevant
indices were assessed. Heavy metal level, histopathology, and
biochemical analyses of the organs including oxidative stress
parameters such as MDA, SOD, CAT, were assayed. The levels
of NO and total thiol group were also assessed.
The significantly high level of toxic metals observed in this
study is secondary to sequestration in the tissues. Significant
bioaccumulation which was seen in the first 14 days of expo-
sure both in the liver and kidney was not accompanied by a
corresponding magnitude of bioaccumulation for the follow-
ing 14 days of exposure (for the 28-day groups) despite the
maintenance of constant effusion rate of exposure throughout
the experiment. This is consistent with all the metals analyzed.
Huge perfusion necessitated by the functions of the organs
predispose them to xenobiotic content of the blood including
heavy metals. This may answer for the tissue bioaccumulation
observed in this study. Heavy metals are hardly metabolized
in the body (Cooper et al. 2017). They prevent the formation
of biochemical complexes required for physiological func-
tions by competing for the binding sites on transport proteins
thereby displacing ligands (Singh et al. 2011).
The weight of liver and kidney were significantly increased
when compared with their respective controls. The last 14 days
of the 28-day group differ from that seen in the first 14 days of
exposure (14-day group). This condition may be indicative of
bioaccumulation of heavy metals and local tissue response to
cement dust. This has been reported to induce oxidative stress
(Tanju and Madhuri 2013; Madejczyk et al. 2015) and as a
precursor of carcinogenesis (Okolie and Osagie 1999). This
observation is in synchrony with the vast majority of literature
information on the organ response to heavy metal intoxication
(Andjelkovic et al. 2019; Dwivedi and Gupta 2020).
There was a general increase in the values of serum
parameters within the first 14 days of exposure to cement
dust. However, the following 14 days of exposure featured
a twist with a percentage decrease in comparison with the
14-day group apart from albumin whose value remains
unchanged for the terminal 14  days. This observation
holds true for the total bilirubin, globulin, and APRI index.
Mnkandla et al. 2019 have demonstrated in snails that tran-
sient exposure to heavy metals may potentiate significant
toxicity whereas a prolonged exposure may induce little
or no deleterious effect. This adaptive response is intrinsic
to most biological systems in toxicological condition and
seems to be significantly demonstrated by the liver when
exposed to hepatotoxic xenobiotics.
Hyperbilirubinemia, as seen in this study may have resulted
from any of the pathological conditions such as neonatal jaun-
dice, excessive hemolysis, genetic errors, or ineffective eryth-
ropoiesis including drugs (Kapitulnik et al. 1975). Earlier, our
laboratory had reported inordinate erythropoiesis following
exposure to cement dust with evidence of increased diapha-
neity of the red cell membrane (Owonikoko et al. 2022); a
condition which may not only prone the cell to hemolysis but
also precedes bilirubin formation. This may be responsible
for the significantly elevated level of bilirubin for the exposed
groups. Aminotranferases and phosphatase are non-invasive
bioindicative enzymes synthesized by the liver. Their serum
levels are significantly altered in conditions of hepatic toxicity/
injuries (Pagana and Pagana 2005) and can therefore be used to
effectively monitor hepatic pathological diagnosis or progno-
sis. In this study, the significant increases in the levels of AST,
ALT, and ALP in the exposed group when compared with the
control, as shown in Table 3, are an indicator of hepatic dys-
function. We observed a significant increase in hepatic fibrosis
tendency as shown by the APRI/Fibrotic index in Fig. 2. It,
therefore, appears that the pathophysiological mechanism of
hepatic pathogenesis in response to cement dust exposure fol-
lows liver fibrosis formation.
The significant increase in the level of MDA in this study
suggests that the exposure to cement dust induces reno-
hepatic lipid peroxidation in the exposed animals when com-
pared with the control. An increase in MDA level has been
well known to generally follow exposure to heavy metals
(Patra et al. 2011). The foregoing particularly holds true for
hepatic and renal tissues in heavy metals mediated patho-
genesis (Andjelkovic et al. 2019).
The significantly reduced levels of renal and hepatic SOD
in this study are indicative that cement dust exposure may
induce oxidative stress not only by inducing MDA produc-
tion but also by depletion of endogenous antioxidant enzyme
capacity. Xenobiotics and particularly co-exposure of heavy
13
 Comparative Clinical Pathology

13
 Comparative Clinical Pathology

◂Fig. 11  A–C Photomicrographs of kidney tissues after inhalatory Figs. 10A–C and 11A–C. Table 8 above shows that cement
exposure to cement dust. Arrows in A and B depict vascular conges- dust induces both intrinsic and idiosyncratic hepatotoxicity
tion. A1 Kidney tissue photomicrograph of rats exposed to cement
dust for 28  days (×  100). A2 Kidney tissue photomicrograph of rats
considering the array of the observed histomorphometry rang-
exposed to cement dust for 28 days (× 400). ­B1: Kidney tissue pho- ing from steatosis, necrosis to vascular congestion. Steatosis
tomicrograph of rats exposed to cement dust for 14 days (× 100). B2 is an early sign of liver injury in response to hepatotoxicant.
Kidney tissue photomicrograph of rats exposed to cement dust for Exposure to cement dust as experimented in this study may
14  days (×  400). C1 Kidney tissue photomicrograph of control rats
(× 100). C2 Kidney tissue photomicrograph of control rats (× 400)
stimulate the appearance of fatty vesicles within the paren-
chymal cellular cytoplasm in the liver lobule. Constituents
of cement dust such as Iron (Fe) have been shown to induce
metals have been severally reported to stimulate hepato-renal hepatic necrosis (Jaishankar et al. 2014). The heavy hepatic
MDA production and deplete antioxidant enzyme status in perfusion of toxic metal-laden blood may have caused these
a variety of experimental animals such as snail (Mnkandla metals to bioaccumulate in the liver and induce chemotaxic
et al. 2019), rats (Yilmaz et al. 2017; Andjelkovic et al. 2019), processes that traumatized hepatocytes causing them to spill
mice (Goc et al. 2019), and fish (Hermenean et al. 2015). their contents. This is well correlated with the significant
In this study, we observed highly significant hepatic and increase in the level of the liver enzymes ALT, AST, and
renal levels of NO in cement dust exposed groups when com- ALP of the 14-day and 28-day exposed groups when com-
pared with the control. Considering the significant manifold pared with the control in Table 3 above. The most significant
increases observable even in the 28-day exposed group, it renal injury observed in this study is inflammation-mediated.
appears that the exposure activated macrophages which stimu- Heavy metals can be transported in the plasma freely or pro-
lated the cytokine-inducible nitric oxide synthase (iNOS) via tein-bound. The latter is conjugated with special antioxidants
the essential cofactor tetrahydrobiopterin (BH4). However,
rather than consider the consequential increase NO bioavail-
ability in the exposed groups as a biomolecular result of the Table 8  Effect of cement dust exposure on reno-hepatic histomorpho-
exposure, we view the condition as a pathophysiological tran- metry
sition to a more deleterious injury. Excess NO secondary to
Histopathological 14-day 28-day Scale Description
iNOS upregulation has been known to exhibit cytotoxicity via changes
activation of the inflammatory cell (neutrophil), potentiates the
synthesis of nitrosamine; a carcinogen (Ribbons et al. 1995; Liver
Ohshima and Bartsch 1994) as well as form extremely toxic Hemorrhagic lesion 2 4 Severe Pericentrilobular
peroxynitrile radical by interacting with superoxides and cul- Inflammation 3 4 Moderate Periportal
minating mutagenesis and DNA damage (Singer et al. 1996; Inflammation 2 2 – Zone 2
Jourd’heuil et al. 1997). Steatosis 3 2 Moderate Macrovesicular
It is noteworthy in this study that the first 14 days expo- Infiltration 4 4 – Inflammatory Cell
sure culminated in higher systemic effect than the following Proliferation 0 3 – Bile Ductless
14 days which precipitated marginal effect compared to the Proliferation 0 2 – Connective Tissue
first 14 days. This is possibly due to the systemic adaptive Necrosis 2 2 – –
adjustment of the experimental animals to the toxic condi- Congestion 3 2 Vascular
tion occasioned by the exposure to cement dust. It is like- Kidney
wise noteworthy that the level of NO response was highly Inflammation 2 3 Moderate Peritubular
significant which points to the pro-inflammatory activity of Inflammation 2 4 Moderate Periglomerular
the exposure to cement dust. Inflammation 2 4 Moderate Perivascular
The significant decrease in the total thiol as observed in Congestion 2 2 Mild Focal Area
this study connotes the antioxidant depleting effect of the dust. The observed histopathological alteration in the hepatic and renal tis-
Asides from inactivating important ions that serve as co-factor sues were assessed using the grade below:
such as Zinc (Zn), heavy metals are known to attack the sulfhy- 0: morphological change absent in histopathological slides from ani-
dryl group (Flora et al. 2007). The depleted level of sulfhydryl mals in a group
as observed in this study may be due to the chelation of the 1: morphological change rarely found in histopathological slides from
animals in a group
heavy metals which reduces their bioavailability.
The bioavailability of heavy metals has been detected in 2: morphological change found in histopathological slides from few
animals in a group
blood (Nwafor et al. 2019) and also bioaccumulated in tis-
3: morphological change common to histopathological slides from
sues (Andjelkovic et al. 2019; Ugbaja et al. 2020) following most animals in the group
cement dust exposure. Cement dust-induced cytoarchitec- 4: morphological change common in histopathological slides from all
tural alteration of the hepatic and renal tissues is as shown in animals in the group

13

such as glutathione and they are released into the blood via the
hepatic or renal pathway. These compounds are reabsorbed
by endocytosis in the proximal convoluted tubule where they
stimulate inflammation or even renal failure (Lentini et al.
2012). This may be the origin of the diffused renal inflam-
mation observed in the exposed groups.
Conclusion
Asides the gastrointestinal tract which connects the external
environment into the internal milieu, the respiratory system
in its handling of gaseous/particulate matter also significantly
mediates the entry of materials including xenobiotics into the
body. Data from this study suggest that experimental inhala-
tion exposure to cement dust induces hepatic and renal inju-
ries in Wistar rats via multiple pathways involving increased
organ weight, tissue heavy metal bioaccumulation, oxidative,
and nitrosative stress mechanisms via induction of lipid per-
oxidation, depletion of antioxidant status and significant nitric
oxide production, and diffuse cytoarchitectural alteration.
Acknowledgements  The authors wish to tremendously acknowledge
the efforts of Prof Kolawole O. Falade of the Department of food Tech-
nology, University of Ibadan, Ibadan, for making some useful equip-
ment available for the purpose of this work. We are equally grateful
to the duo of Messrs Eriki Austin Omokhoa and M. J. Effiong of the
same department for their technical assistance throughout the period.
Data Availability  Should any raw data files be needed in another for-
mat, they are available upon request from the corresponding author.
Compliance with Ethical Standards  Effect of taurine on ethanol-induced oxidative stress in mouse
Funding  This research work was not supported by any source of fund.
Conflict of interest  The authors of this work declare no conflict of interest.
Ethical approval  All procedures performed in studies involving human
participants were in accordance with the ethical standards of the insti-
tutional and/or national research committee and with the 1964 Helsinki
declaration and its later amendments or comparable ethical standards.
Informed consent  Informed consent was obtained from all individual
participants included in the study.
Consent for publication  Consent for publication was obtained for every
individual person’s data included in the study.
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