Drug and Chemical Toxicology

ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/idct20

Protection of CCl4-induced hepatic and renal

damage by linalool

Mohammad Mazani , Lotfollah Rezagholizadeh , Saeedeh Shamsi , Sina

Mahdavifard , Masoud Ojarudi , Ramin Salimnejad & Ahmad Salimi

To cite this article: Mohammad Mazani , Lotfollah Rezagholizadeh , Saeedeh Shamsi , Sina
Mahdavifard , Masoud Ojarudi , Ramin Salimnejad & Ahmad Salimi (2020): Protection of CCl4-
induced hepatic and renal damage by linalool, Drug and Chemical Toxicology

To link to this article: https://doi.org/10.1080/01480545.2020.1792487

Published online: 13 Jul 2020.

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DRUG AND CHEMICAL TOXICOLOGY
https://doi.org/10.1080/01480545.2020.1792487

RESEARCH ARTICLE

Protection of CCl4-induced hepatic and renal damage by linalool

Mohammad Mazania
, Lotfollah Rezagholizadeha
, Saeedeh Shamsib, Sina Mahdavifarda , Masoud
Ojarudic, Ramin Salimnejadd and Ahmad Salimib
a
Department of Biochemistry, School of Medicine, Ardabil University of Medical Sciences, Ardabil, Iran; bDepartment of Pharmacology and
Toxicology, School of Pharmacy, Ardabil University of Medical Sciences, Ardabil, Iran; cDepartment of Biochemistry, Faculty of Sciences,
Payame Noor University, Tehran, Iran; dDepartment of Anatomical Sciences and Pathology, School of Medicine, Ardabil University of Medical
Sciences, Ardabil, Iran

ABSTRACT ARTICLE HISTORY

The aim of the current study is to determine the protective and therapeutic effects of linalool against Received 7 April 2020
carbon tetrachloride (CCl4)-induced hepatoxicity and nephrotoxicity. Six-week-old male Wistar rats were Revised 10 June 2020
divided into five groups: Control group (a regular diet); CCl4 group (1 ml/kg dissolved in olive oil, intra- Accepted 22 June 2020
peritoneally at 14th day); pretreatment group (25 mg/kg linalool daily þ CCl4 14thday); post-treatment
KEYWORDS
group (25 mg/kg linalool 2, 6, 24, and 48 h after the injection of CCl4 at 14th day); and linalool group Linalool; hepatotoxicity;
(25 mg/kg linalool daily, orally). All animals were sacrificed, tissue and blood samples were collected to nephrotoxicity; natural
analysis. Administration of CCl4 resulted in a marked increase in hepatic (aspartate aminotransferase, compounds; oxidative stress
alanine transaminase, and alkaline phosphatase) and renal (blood urea nitrogen and creatinine)
markers. Also, CCl4 resulted in pathological damages, a significant increase in the concentration of
malondialdehyde , tumor necrosis factor-alpha, and Interleukin 6 , expression of nuclear factor kappa-
light-chain-enhancer of activated B cells and a significant decrease in the levels of serum total protein,
serum albumin, and antioxidants. However, in pretreatment and post treatment groups, linalool signifi-
cantly inhibited CCl4- induced hepatic and nephric damages. These results demonstrate that linalool
has protective and therapeutic effects in an in vitro model of CCL4-induced hepatic and nephric dam-
age, proposing linalool as a potential therapeutic agent against chemical and drug induced hepatotox-
icity and nephrotoxicity.

Introduction and kidney damages. Moreover, these medicinal risks, liver

and kidney injuries accompany exposure to different chemi-
The liver and kidneys are main organs affected by drug and
cals in the environment or workplace. Still chemicals such as
chemical toxicity (Zhang et al. 2007). The liver is the main
food contaminants that harm these excretory organs are con-
organ where exogenous and endogenous chemicals are
sumed (Burcham 2014). Therefore, protecting the liver and
metabolized and finally excreted (Almazroo et al. 2017). As a kidney against these agents is inevitable. Natural compounds
consequence, hepatocytes (liver cells) are exposed to remark- found in plants have long been shown to play an important
able concentrations of these chemicals and drugs, which can role in inhibiting liver and kidney toxicity (Nale et al. 2012).
lead to cell death, hepatoxicity, liver dysfunction, and even Herbal medicines have a long history over than 7000 years
organ failure (Russmann et al. 2009). The kidney has a key in the treatment of many diseases. Using natural compounds
role in the regulation of acid-base balance, electrolyte com- to treat diseases mainly in the developing countries, is still
position, and extracellular fluid volume, and in the excretion the pillar of near 75–80% of the treatment in the world (Ekor
of metabolic wastes (Raghavendra and Vidya 2013). 2014). Because of better compatibility with the human body,
Moreover, the kidney synthesizes and releases hormones, and lesser side effects and better cultural acceptability, these
metabolizes vitamin D3 to the active form (Gekle 2017). compounds are used frequently for primary health care (Ekor
Therefore, the functional integrity of the mammalian liver 2014). Also, a major increase in their use in the developed
and kidneys is vital to total body homeostasis (Burcham countries have seen in the last few years. Nowadays, there is
2014). A toxic insult to the liver and kidney could disrupt any a bipolarized market for the active compounds: those natural
or all of these activities and could have profound effects on ingredients that are requested by patients and those chem-
mammalians (Zhang et al. 2012). Therefore, the liver and the ically produced by the pharmaceutical companies (Atanasov
kidneys are more commonly involved in overt organ toxicity et al. 2015). Many of these agents can have ameliorative
than most other tissues (Zhang et al. 2012). Accidental or effects against drugs and chemicals toxicities (Abdel-Daim
deliberate toxicity with drugs can cause life-threatening liver et al. 2016). One of these natural products is linalool (LIN),

CONTACT Ahmad Salimi [email protected], [email protected] Department of Pharmacology and Toxicology, School of Pharmacy, Ardabil
University of Medical Sciences, P.O. Box: 56189-53141, Ardabil, Iran

These authors contributed equally to this study.
ß 2020 Informa UK Limited, trading as Taylor & Francis Group
2 M. MAZANI ET AL.

which found in essential oils of plants such as mint, basil, purchased from R&D Systems Europe, Ltd. (Abingdon, UK).
and coriander. Linalool has several pharmacological effects Linalool (3,7-dimethyl-1, 6-octadien-3-ol) with a purity of
including antibacterial, antimicrobial, anti-inflammatory, anti- about 99% and CAS number 78-70-6 was purchased from
viral, anesthetic, and analgesic effects (Sharifi et al. 2017). Sigma-Aldrich (St Louis, MO). Linalool was freshly prepared
Previous studies showed that linalool, is a competitive antag- before use and dissolved in normal saline 0.9% wt/vol.
onist of N-methyl d-aspartate (NMDA) receptors and produ-
ces analgesic properties via brain opioids when it inhibits
NMDA receptor activity (Batista et al. 2008). In addition, the Animals
hypolipidemic, antihyperglycemic, and antioxidant properties Male Wistar rats weighing 200 ± 20 g was purchased from the
of linalool, its preventive effect on proteinuria and its thera- Baquatollah University of Medical Sciences (Tehran, Iran) and
peutic effect on kidney function were evidenced. It was sug- maintained under a standard dark-light cycle (lights on
gested that some monoterpenes obtained from plants between 7:00and 7:00am) at room temperature (22 ± 2  C).
inhibited and decrease tumor growth. Therefore, recent These conditions were maintained constantly throughout the
investigations have focused on the chemotherapeutic or experiments. This study was approved by the Ethics
chemo-preventive potential of linalool (Kamatou and Viljoen Committee at the Ardabil University of Medical Sciences with
2008). A recent study has shown that linalool has protective ethics codes IR.ARUMS.REC.1397.120 and performed strictly in
effect against glutamate-induced oxidative stress and excito- accordance with institutional and international guide for ani-
toxity (Sabogal-Guaqueta et al. 2019). Oner et al. (2019) mal care.
proved that administration linalool with doxorubicin can
remove cardiomyopathy symptom induced by this drug. It
has been reported that the plasma concentration of linalool Experimental design
reaches about 10 min at the first minor peak, the second Thirty male Wistar rats weighing 200 ± 20 g, aged eight
major peak reaches in about 40 min, and then the linalool weeks, were selected randomly and given free access to
concentration in the plasma decreased rapidly (Shi water and food. After two weeks of acclimation, the animals
et al. 2016). were assigned for experimental procedures. The animals
Carbon tetrachloride (CCl4) is widely used as a model for were divided into five subgroups: controls (normal rats n ¼ 6)
induction of liver and kidney damages in rats to investigate gained free access to food and water; the CCl4 group (n ¼ 6)
potential therapeutic effect of new agents (Suzuki et al. was given 1 ml/kg of CCl4, dissolved in olive oil, through
2015). This chemical is metabolized to active metabolites intraperitoneally at 14th day; the pretreatment group (25 g
such as trichloromethyl-free radicals and peroxyl radical by mg/kg LIN for 14 days plus single dose of 1 mg/kg CCl4); the
the cytochrome P450 enzymes, which then initiate oxidative post-treatment group (25 mg/kg LIN 2, 6, 24, and 48 h after
damages, cell death, and inflammation (Dai et al. 2018a). the injection of CCl4 at 14th day), and the LIN group was
Whereas, linalool has previously been shown to exhibit anti- given normal diet and administered LIN orally (25 mg/kg) for
inflammatory and antioxidant properties and to the best of 14 days. The dose of CCl4 and LIN was determined based on
our knowledge, there is no literature on the effects of linalool previous studies (Dai et al. 2018a, Sabogal-Guaqueta et al.
on hepatoxicity and nephrotoxicity. The aim of the present 2019). At the end of the experimental procedures, all animals
study was to determine the protective and therapeutic were weighed and then anesthetized using a combination of
effects of linalool, on tetrachloride-induced hepatoxicity and xylazine (10 mg/kg) and ketamine (10 mg/kg) administered
nephrotoxicity in rats using histopathologic, genes analysis via intraperitoneal (i.p.) injections. Liver and kidney tissues
and biochemical methods. were taken for RNA extraction and gene expression (reverse
transcription polymerase chain reaction, RT-PCR) as well as
Materials and methods for histopathology and antioxidant capacity.

Chemicals and kits

Serum extraction and chemistry analysis
Hydrogen peroxide, methanol, thiobarbituric acid, bovine
serum albumin (BSA), Coomassie blue, CCl4, iron sulfate, ferric Blood was collected directly from their heart using hepari-
chloride, sodium acetate, ketamine, xylazine, and butanol nized capillary tubes. The blood samples were centrifuged at
were purchased from Merck Company (Darmstadt, Germany). 3000 rpm for 10 min and the supernatants (serums) were
The Wistar albino rats were purchased from the Baquatollah transferred into 0.5 ml vials and were frozen at 80  C for the
University of Medical Sciences (Tehran, Iran). The kits for subsequent biochemical tests and the analysis of inflamma-
superoxide dismutase (SOD) and glutathione peroxidase tory factors. Serum creatinine, BUN, bilirubin, albumin, total
(GPx) were purchased from Randox Company (Crumlin, UK). protein, ALT, AST, and ALP was measured using commercially
The kits for assaying of aspartate aminotransferase (AST), ala- available kits based on spectrophotometric analysis.
nine transaminase (ALT), alkaline phosphatase (ALP), blood
urea nitrogen (BUN), creatinine, bilirubin, serum total protein,
Determination of liver and kidney antioxidant activity
and serum albumin levels were purchased from Pars-Azmoon
Company (Tehran, Iran). The kits for assaying tumor necrosis For malondialdehyde (MDA), total antioxidant capacity (TAC),
factor-alpha (TNF-a) and interleukin-6 (IL-6) levels were superoxide dismutase (SOD), catalase (CAT), glutathione
 DRUG AND CHEMICAL TOXICOLOGY 3

peroxidase (GPx), catalase activity measurements, 200 mg of protocols. Two micrograms of total RNA were reversely tran-
liver or kidney slices was homogenized in 2 ml of homogen- scribed into cDNA by using SMOBIO strand cDNA synthesis kit
ization buffer (50 mM potassium phosphate buffer; PBS, pH (lot No #CHRP 051901430-8, Taiwan), according to the man-
7.4). The homogenized tissues were centrifuged at ufacturer’s instructions. Quantitative RT- PCR was carried out
10 000 rpm for 2 min. The supernatants were centrifuged using TB Green Premix Ex TaqTM II (cat#RR820Q, TaKaRa, China)
again at 12 000 rpm for 20 min to obtain the pure superna- kit on LightCyclerV R 96 System (Roche Applied Science,
tants. The final obtained supernatants were used for the Mannheim, Germany). Actb was applied as housekeeping gene
measurement of the antioxidant activity. The activities of for normalization of gene expression data. RT- PCR primer
SOD, CAT, GPx, and TAC were determined using commercially sequences were as follows, Rela: 50 -CCTGTCTGCACCTGTTCCAA-
available kits based on spectrophotometric analysis by 30 (forward), and 50 -ACTCCTGGGTCTGTGTTGTT-30 (reverse), Actb:
StatFax 3000 ELISA reader . Results of SOD, CAT, GPx, and 50 -GGAGAA GATTTGGCACCACACT-30 (forward), and 50 -
TAC activities were calculated according to the manufac- 0
CGGTTGGCCTTA GGGTTCAGA-3 (reverse). The comparative CT
turer’s instructions. method was performed to measure the relative expression lev-
els of Rela in liver samples. All qRT-PCR data were normalized
Liver and kidney histopathology after subtracting the CT values of Rela from that of Actb as an
internal control (2-DCT method, DCT ¼ CTRela – CtActb). Each
At the end of the experimental procedures, livers and kidneys measurement was performed in triplicate.
of all experimental animals were removed. The removed tis-
sues were fixed in a buffered neutral formalin solution (10%).
Fixed tissues were processed routinely including washing, Data analysis
dehydration, clearing, paraffin embedding, casting, and were The results were analyzed using Graph Pad Prism (version 5,
sectioned to 4–5 mm thickness for using in hematoxylin and Graph Pad Software Inc., La Jolla, CA). Results are presented as
eosin (H&E) staining. To examine the histological alterations mean ± standard deviation for six independent rats per each
12 images were selected randomly in each sample and the group. The statistical significance of the differences between
histological index was calculated semi-quantitatively using a
groups was assessed using one-way analysis of variance, post-
scale of 0–3: (0) none, (1) mild, (2) moderate, and (3) severe
hoc test, and Fisher’s least significant difference (LSD) test.
damage. The investigated damages include degenerated
cells, necrotic cells, vacuolation, cell detachment, and
lymphocyte infiltration (Peng et al. 2018). Results
Protective and therapeutic effect of LIN on CCl4
Real time quantitative RT-PCR analysis -induced hepatic and renal histopathological changes
Total RNA was extracted from liver samples with RNX- Plus (Cat Hepatic changes in control and LIN-administrated showed
No. EX 6101, SINACLON, Tehran, Iran) according to the kit normal hepatic architecture illustrated by hepatic lobule with

Figure 1. (a) Photomicrograph of the liver of normal control rats shows a normal hepatic architecture represented by hepatic lobule with a thin walled central vein.
(b) Photomicrograph of the liver of treated rats with CCl4 shows restoration of normal hepatic architecture with disappearance of fat droplets from hepatocyte cyto-
plasm; congestion of the central vein; lymphocytic infiltration; and hydropic degeneration in the hepatocytes, (arrows) and regeneration of hepatic parenchyma. (c)
Photomicrograph of the liver of the CCl4 þ Lin group shows disappearance of most of the degenerative changes. (d) Photomicrograph of the liver of Lin group
shows normal architecture as that of the control group (H&E, 400).
4 M. MAZANI ET AL.

a thin walled central vein and normal hepatic cords radiating restored such alterations to normal levels and LIN has no toxic
toward the periphery alternating with hepatic sinusoids. In effects on hepatic, and renal function parameters.
the CCl4 group, the livers showed hydropic degeneration in
the hepatocytes, lymphocytic infiltration, and congestion of
Protective and therapeutic effect of LIN on CCl4
the central vein. The liver of the protective and post treat-
-induced changes in enzymatic antioxidant defense
ment group (CCl4 plus LIN) demonstrated disappearance of
systems and MDA content of liver and kidney
the degenerative changes in hepatocytes except few hepatic
cells and edema in the central vein showed hydropic degen- As shown in the Table 1, in the CCl4 group, the activity of
eration (Figure 1(a–d)). The normal kidney in control and LIN- serum TAC, CAT, SOD, and GPx was significantly (p < 0.001)
administered rats showed a normal renal architecture repre- reduced as compared to the control group and LIN group. In
sented by renal tubules and renal corpuscles (Figure 2(a–d)). the LIN þ CCl4 group, linalool could significantly (p < 0.001)
In the CCl4-administered group, renal histology showed con- ameliorate the activity of these enzymes compared to the
gestion of the renal blood vessels, degeneration in the cells CCl4 group. Also, administration of linalool (25 mg/kg) alone
of the proximal and distal convoluted tubules and vacuol- for 14 days caused a significant (p < 0.001) increase in the
ation of the renal tubules (Figure 2). In the protective and activity of the enzymes as compared to the control group.
posttreatment group, LIN induced recovery and regeneration
in renal structure from biohazards of CCl4 (Figure 2(a–d)).
Correspondingly, histological scores were significantly
decreased to 2.3 and 1.8 in liver and kidney tissues, respect-
ively compared to CCl4-treated group (Figure 3).

Protective and therapeutic effect of LIN on CCl4-induced

changes in serum biochemical profiles, hepatic, and
renal function tests
The results in Figure 4(a–d);liver) and Figure 4(e–h);kidney) show
that oral administration of LIN for 14 days increased the serum
levels of ALT, AST, ALP, BUN, creatinine, albumin, bilirubin, and
total protein in serum of CCl4 administered rats compared to
control and LIN administered rats. Co-administration of LIN
together with CCl4 normalized such alterations. Moreover, LIN- Figure 3. Semi-quantitative analysis of protective and therapeutic effects of Lin
against CCl4 induced the liver and kidney damages. The histological index was calcu-
administered rats did not show increase in serum levels of ALT, lated semi-quantitatively using a scale of 0–3: (0) none, (1) mild, (2) moderate, and
AST, ALP, BUN, creatinine, albumin, bilirubin, and total protein. (3) severe damage. The results are expressed as mean ± standard deviation (SD).



p < 0.05 compared with NC group; †p < 0.05 compared with CCl4 group.
These data indicated that LIN co-administration with CCl4

Figure 2. (a and d) Photomicrograph of the kidney of normal control and Lin-administered rats shows normal renal architecture represented by renal tubules and
renal corpuscles. (b) Photomicrograph of the kidney of CCL4-administered rats shows congestion of the renal blood vessels, vacuolation of the renal tubules, and
degeneration of the cells of the proximal and distal convoluted tubules. (c) Photomicrograph of the kidney of CCl4 þ Lin group shows recovery of the renal tissue
to normal structure (H&E, 400).
 DRUG AND CHEMICAL TOXICOLOGY 5

Figure 4. Protective and therapeutic effects of Lin on CCl4 induce changes in the liver and kidney function with evaluation of (a) AST, (b) ALT , (c) ALP, (d) liver
albumin, (e) Creatinine, (f) BUN, (g) total bilirubin, and (h) kidney albumin using commercially available kits. The results are expressed as mean ± standard deviation
(SD) for the rats in each group.


Shows the significance of the differences relative to the normal control group (p < 0.001). ††† Shows the significance of the dif-
ferences relative to CCl4 group (p < 0.001).

Protective and therapeutic effects of LIN on CCl4

higher IL-6, and TNF-a concentrations in liver and kidney tis-
-induced TNF-a and IL-6 productions
sues as compared to the control group. However, pretreat-
We investigated whether LIN decreases the production of the ment with 25 mg/kg LIN caused significantly (p < 0.05) lower
pro-inflammatory cytokines: TNF-a and IL-6 induced by CCl4. concentrations of these inflammatory in these liver and kid-
The toxic effect of CCl4 has resulted in significant (p < 0.05) ney tissues than those not treated (Figure 5(a,b)).
6 M. MAZANI ET AL.

Figure 5. Protective and therapeutic effects of Lin on CCl4 induce changes in the production of TNF-a and IL-6 in liver tissue of Wistar rats using commercially avail-
able kits. The results are expressed as mean ± standard deviation (SD) for the rats in each group.


Shows the significance of the differences relative to the normal
control group (p < 0.001). ††† Shows the significance of the differences relative to CCl4 group (p < 0.001).

Table 1. Protective and therapeutic effect of linalool (Lin) on CCL4 induced changes in hepatic and renal MDA, TAC, and antioxidant enzymes (SOD, GPX, and
CAT) activity.
Experimental groups NC Lin CCL4 Pre-Lin Post-Lin
Liver
MDA (nmol/mg protein) 0.10 ± 0.014 0.11 ± 0.012 0.34 ± 0.027


0.10 ± 0.013 ††† 0.13 ± 0.012†††
TAC (mmol/liter) 0.41 ± 0.05 0.41 ± 0.05 0.22±.04


0.39 ± 0.05††† 0.34 ± 0.04†††
SOD (U/mg protein) 11.05 ± 0.54 11.07 ± 0.54 7.8 5 ± 0.15


8.55 ± 0.17†† 7.92 ± 0.49
GPx (U/mg protein) 4.69 ± 0.44 4.78 ± 0.40 3.39 ± 0.49


4.58 ± 0.40††† 3.83 ± 0.40
CAT (U/mg protein) 86.17 ± 10.71 89.15 ± 10.66 41.84 ± 14.00


66.88 ± 10.81†† 58.87 ± 15.66†
Kidney
MDA (nmol/mg protein) 2.2 ± 0.122 1.7 ± 0.20 24 2.2 ± 2.1


13.06 ± 1.11††† 9.9 ± 1.8†††
TAC (mmol/liter) 414.8 ± 43 409.03 ± 45 263.8 ± 39


356.2 ± 47††† 371. 7 ± 41†††
The results presented in the table are expressed as mean ± standard deviation (SD) for the rats in each group.



Shows the significance of the differences relative to the normal control group (p < 0.001).
†, ††, and ††† Show the significance of the differences to CCl4 group (p < 0.05, 0.01, and 0.001, respectively).

Protective and therapeutic effect of LIN on CCl4 Solvents are highly lipophilic, which rapidly absorb and dis-
-induced changes in hepatic nuclear factor kappa-light- tribute in the body (Fiserova-Bergerova 1985). The presence
chain-enhancer of activated B cells (NF-jB) expression of the enzymatic systems necessary for solvent metabolism
and the high levels of transition metals found in the liver
Administration of CCl4 with single dose increased mRNA
and kidney have led to the oxidative stress in toxicity these
expression of NF-jB (Figure 6). LIN alone decreased the agents (Brautbar and Williams II 2002, Al-Ghamdi et al. 2003).
expression of NF-jB. Co-administration of LIN and CCl4 nor- A number of enzymes might be responsible for an associ-
malized such increase in NF-jB expression reported in CCl4 ation between and oxidative stress and solvent exposure
group. These results indicate that co-administration of LIN (Costa et al. 2006). One of the main enzyme systems in acti-
with CCl4 decreases NF-jB. vation of solvent to reactive metabolite is cytochrome P450
enzymes which use molecular oxygen to oxidize some
Discussion organic solvents (Furge and Guengerich 2006). This oxidation
of solvents produces reactive oxygen species, especially very
Acute and chronic exposure to some organic solvents is reactive intermediate of hydroxyl radical. In turn, hydroxyl
known to cause hepatotoxicity and nephrotoxicity radicals react with many cellular components such as pro-
(Malaguarnera et al. 2012). In the past of several decades, oxi- teins, polyunsaturated fatty acids, nucleic acids.
dative stress has been found to be the man mechanism of Concentrations of cytochrome P450 are high in hepatic and
action of a number of some solvent (Kum et al. 2007). renal tissue, and can be induced by xenobiotics, including
 DRUG AND CHEMICAL TOXICOLOGY 7

body becomes overwhelmed and lead to toxicity responses

such adaptive, inflammatory, reparative and injurious proc-
esses (Kehrer and Klotz 2015). Oxidative stress has been con-
sidered to be closely associated with nuclear factor (NF)-jB
(Niederberger and Geisslinger 2008). A vicious cycle has been
reported between the oxidative stress and NF-jB pathway.
For the activation of the NF-jB signaling pathway, oxidative
stress is crucial, it results in the aggravation of oxidative
stress (Morgan and Liu 2011).Our results at gen, molecular
cellular and tissue levels showed that exposure with indus-
trial solvent CCL4 caused all toxicity responses mentioned in
above and created a state of oxidative stress condition in
these two organs. Today, antioxidants as potential medical
countermeasures are used for toxic industrial chemicals
(McElroy and Day 2016). Many of these agents produce anti-
oxidant properties indirectly or even paradoxically
(Devasagayam et al. 2004). Direct acting antioxidants that
Figure 6. Protective and therapeutic effects of Lin on CCl4 induce changes in scavenge reactive species may often generate a reactive
the expression of NF-kB in liver tissue of Wistar rats using semi-quantitative RT- product (Winterbourn 2008). This condition happens with
PCR analysis. RNA was extracted and reverse transcribed and RT-PCR analysis
was carried out NF-kB expression as described in materials and methods. The SOD that dismutate superoxide and produces hydrogen per-
results are expressed as mean ± standard deviation (SD) for the rats in each oxide (Ighodaro and Akinloye 2018). Previous studies have
group.


Shows the significance of the differences relative to the normal con- demonstrated the high antioxidant potential of linalool on
trol group (p < 0.001). ††† Shows the significance of the differences relative to
CCl4 group (p < 0.001). reactive species scavenging in comparison with ascorbic acid
(Seol et al. 2016). These studies confirmed that the linalool
organic solvents (Pelkonen et al. 2008). Therefore, these can be used in the synthesis of several type of compound
enzymes play an important role in transforming organic sol- with ability to act as antioxidant (Jabir et al. 2018). They sug-
vents that have exposed with the liver and kidney. Finally, gested that linalool as a medicine drug can be used in the
this can lead to oxidative stress with the production of free treating several types disease related to oxidative stress (Seol
radicals and lipid peroxidation in the hepatic and renal tis- et al. 2016). Our results confirmed that the linalool has pro-
sues. CCl4 as an organic solvent and agent in induction of tective and therapeutic effect against CCl4-induced hepatic
liver and kidney damage mode, is activated by cytochrome and liver damages.
P450, to form the trichloromethyl radical, CCl3
. Exposure to drugs, chemicals, mechanical, and thermal
Trichloromethyl radical can bind to nucleic acids, proteins, injuries cause inflammation as a tightly regulated immune-
and lipids. Our result in the current study showed that expos- protective response to combat these agents (Kidd and Urban
ure with CCl4 led to oxidative stress and lipid peroxidation in 2001). Our results showed that CCL4 induces the production
hepatic and renal tissues. These data are consistent with pre- of pro-inflammatory cytokines such as IL-6, and TNF-a from
viously published data examining effect of CCl4 on liver and liver and kidney tissues which these data are consistent with
kidneys (Muriel 1998, Tirkey et al. 2005). Due to the import- previously published data in this regard (Dai et al. 2018b).
ance of the cytochrome P450 enzymes in activation of xeno- Inhibition of inflammation is always one of the most
biotics, assessment of the potential of compounds which important actions in controlling the toxicity effect of these
reversibly inhibit cytochrome P450 enzymes can be an factors. Nonsteroidal anti-inflammatory drugs (NSAIDs) are
important strategy in reduction of toxicity. Previous studies used to suppress prostaglandin E2 (PGE2) production
have been suggested that linalool is weak competitive inhib- through the inhibition of cyclooxygenase-2 (COX-2). There is
ition of cytochrome P450 enzymes in rat liver microsomes a strong evidence that have shown, the prolong exposure to
(Ganzera et al. 2006, Noskova et al. 2016), therefore, this com- this drug class has been associated with serious and some-
pound can reduce the toxicity of substances that exert their times life-threatening side effects (Mbonye et al. 2008).
toxicity from this pathway. In our study, pretreatment and Hence it is necessary to find out alternative anti-inflammatory
post treatment with linalool showed a significant decrease in agents with comparative or more efficacy than NSAIDs but
CCl4-induced oxidative paraments. Probably inhibition of the with fewer side effects. All kinds of inflammatory conditions
cytochrome P450 by linalool could be effective in creating have been treated with the natural products. These natural
this effect. products later become the counter stones for producing that
There is powerful evidence that reactive species (RS) is main ani-inflammatory drug such as Aspirin. Therefore, the
involved in oxidative stress as a main mechanism by which exploration in the natural products to find out the bioactive
industrial solvents induce liver and kidney damages (Jyothi compounds can be quite promising and resulting in the dis-
et al. 2012). Due to RS high reactivity, these intermediates covery of numinous secondary metabolites with extra ordin-
prone to cause damage to any type of molecule within the ary bioactivities (Attiq et al. 2018). Our results showed that
cell (Bergamini et al. 2004). Due to oxidative damages to the linalool can inhibit the expression and production against
cellular components, the antioxidant defense in the human CCl4-induced induction of inflammatory mediators.
8 M. MAZANI ET AL.

Conclusion Brautbar, N. and Williams, II, J., 2002. Industrial solvents and liver toxicity:
risk assessment, risk factors and mechanisms. International Journal of
In conclusion, the present study was designed to yield quan- Hygiene and Environmental Health, 205 (6), 479–491.
titative evidence of the protective and therapeutic properties Burcham, P. C., 2014. Target-organ toxicity: liver and kidney. In: P. C.
Burcham, editor. An introduction to toxicology. London: Springer.
of linalool against CCl4-induced hepatic and renal damages.
Costa, C., et al., 2006. In vitro evaluation of oxidative damage from
The positive effects of linalool such reduction of oxidative organic solvent vapours on human skin. Toxicology in Vitro, 20 (3),
stress and expression of anti-inflammatory mediators and 324–331.
enhancement of antioxidant defense were proved in this Dai, C., et al., 2018a. Chloroquine ameliorates carbon tetrachloride-induced
study. However, further studies are needed to clarify the acute liver injury in mice via the concomitant inhibition of inflammation
and induction of apoptosis. Cell Death & Disease, 9 (12), 1–13.
mechanism of the protective and therapeutic effect of lina- Dai, C., et al., 2018b. Chloroquine ameliorates carbon tetrachloride-induced
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constituents on human cytochrome P450 enzymes. Life Sciences, 78
The authors declare that they have no conflict of interest. (8), 856–861.
Gekle, M., 2017. Kidney and aging—a narrative review. Experimental
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Funding Ighodaro, O. and Akinloye, O., 2018. First line defence antioxidants-super-
oxide dismutase (SOD), catalase (CAT) and glutathione peroxidase
This study was supported by Ardabil University of Medical Sciences, (GPX): their fundamental role in the entire antioxidant defence grid.
Deputy of Research with ethics code IR.ARUMS.REC.1398.281. Alexandria Journal of Medicine, 54 (4), 287–293.
Jabir, M.S., Taha, A.A., and Sahib, U.I., 2018. Antioxidant activity of lina-
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and antioxidants in liver and kidney of rats. Indian Journal of
Mohammad Mazani http://orcid.org/0000-0001-9537-8572
Biochemistry and Biophysics, 49, 279–284.
Lotfollah Rezagholizadeh http://orcid.org/0000-0003-3175-1360
Kamatou, G.P. and Viljoen, A.M., 2008. Linalool–a review of a biologically
Sina Mahdavifard http://orcid.org/0000-0001-9878-2984
active compound of commercial importance. Natural Product
Ramin Salimnejad http://orcid.org/0000-0002-7437-502X
Communications, 3 (7), 1934578X0800300.
Ahmad Salimi http://orcid.org/0000-0003-3026-6398
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