The anticancer activity of five species of Artemisia on Hep2 and HepG2 cell lines

Abstract

It has been reported that several Artemisia species (Astraceae) possess cytotoxic activity against different human cell lines. In this study, the toxicity of the A. kulbadica, A. sieberi, A. turanica, A. santolina and A. diffusa against human Caucasian hepatocyte carcinoma (HepG-2) and human Caucasian larynx carcinoma (Hep-2) cell lines have been investigated. These plants were collected from Khorasan province, northeast of Iran. Different concentrations (200, 400, 600, 800, 1000, 1200, 1600 and 3200 μ g/mL) of ethanol extract of each sample were prepared. The cytotoxic effects of these concentrations against two human tumor cell lines Hep2 and HepG2 were determined by quantitative MTT assay. Results showed concentration- and time-dependent toxicity. In all extracts, toxic effects were significantly higher on HepG2 cells compared with Hep2 cells. HepG2 cells are rich in phase I and phase II metabolic enzymes and it is probable that metabolic activation of some active ingredients of the extracts were converted to more toxic metabolites and caused more toxicity in these cells.

Full text

Pharmacologyonline 3: 327-339 (2009) Emami et al.
327
The Anticancer Activity of Five Species of Artemisia on
Hep2 and HepG2 Cell Lines
Seyed Ahmad Emami, Nasser Vahdati-Mashhadian*, Remisa Vosough and Mohammad
Bagher Oghazian
Drug Research Centre, School of Pharmacy, Mashhad University of Medical Sciences
(MUMS), Mashhad, I R Iran
Summary
It has been reported that several Artemisia species (Astraceae) possess cytotoxic activity
against different human cell lines. In this study, the toxicity of the A. kulbadica, A. sieberi,
A. turanica, A. santolina and A. diffusa against human Caucasian hepatocyte carcinoma
(HepG-2) and human Caucasian larynx carcinoma (Hep-2) cell lines have been investigated.
These plants were collected from Khorasan province, northeast of Iran. Different
concentrations (200, 400, 600, 800, 1000, 1200, 1600 and 3200 µg/mL) of ethanol extract of
each sample were prepared. The cytotoxic effects of these concentrations against two human
tumor cell lines Hep2 and HepG2 were determined by quantitative MTT assay.
Results showed concentration- and time-dependent toxicity. In all extracts, toxic effects were
significantly higher on HepG2 cells compared with Hep2 cells. HepG2 cells are rich in phase I
and phase II metabolic enzymes and it is probable that metabolic activation of some active
ingredients of the extracts were converted to more toxic metabolites and caused more toxicity in
these cells.
Keywords: Hep2, HepG2, Artemisia spp., Hepatotoxicity, MTT assay.
* Author of correspondence:
Nasser Vahdati-Mashhadian
Drug research center
School of Pharmacy
Mashhad University of Medical Sciences
Mashhad, 91775-1365, Iran
E-mail: vahdatin@mums.ac.ir
Fax No. +98-511-8823251

Pharmacologyonline 3: 327-339 (2009) Emami et al.
328
Introduction
The genus Artemisia is one of the largest and most widely distributed of the nearly 100 genera
in the tribe Anthemideae of the Asterceae (Compositae). This is a large and heterogeneous
genus, numbering over 400 species distributed mainly in the temperate zone of Europe, Asia and
North America. These species are Perennial, biennial and annual herbs or small shrubs,
frequently aromatic. Leaves are alternate, capitula small, usually pendent, racemose, paniculate
or capitate inflorescences, rarely solitary. Involucral bracts stand in few rows, receptacle flat to
hemispherical, without scales, sometimes hirsute. Florets are all tubular, Achenes obvoid,
subterete or compressed, smooth, finely striate or 2-ribbed; pappus absent or sometimes a small
scarious ring (1-3).
The genus in Iran has about 34 species which two of them are endemic to the country (3-
6)..These plants contain monoterpenes, sesquiterpenes, sesquiterpene lactones, flovonoides,
coumarins, sterols, polyacetylenes etc. (1).
Artemisia species have been shown to have wide range of pharmacological and toxicological
effects, including antimalarial (7, 8), cytotoxic (9), antifungal (10-12) and antioxidant (12-14)
activities.
In this study, the anticancer activity of five species of the genus against two human cancer cell
lines (Hep2 and HepG2) was investigated.
Materials and Methods
Plant material
Five species of Artemisia were collected from different parts of Iran (Table 1). Dr. V.
Mozaffarian, Research Institute of Forest and Rangelands, Ministry of Jahad-E-Agriculture Iran,
confirmed the identity of the plants. Voucher specimens of the species have been deposited in the
Herbarium of National Botanical Garden of Iran (TARI).
Table 1- Characteristics of collected Artemisia species
Artemisia species Location Collection
time
A. diffusa Krasch.
ex Poljak. Mazdavand, Khorasan Razavi province
(height 900 m) Aug. 19, 2007
A. kulbadica
Boiss. & Buhse Islamabad, Shahrabad road, Northern
Khorasan province (height 907 m) Aug. 5, 2007
A. santolina
Schrenk Daghe Akbar, Southern Khorasan province
(height 1460 m) Sep. 20, 2007
A. sieberi Besser Samie Abad, Khorasan Razavi province
(height 909 m) Sep. 15, 2007
A. turanica Krash Samie Abad, Khorasan Razavi province
(height 808 m) Sep. 15, 2007

Pharmacologyonline 3: 327-339 (2009) Emami et al.
329
Cell cultures and treatments
HepG-2 and Hep-2 cells were purchased from Pasteur Institute Collection of Cell Cultures,
Tehran, Iran and were cultured in DMEM (Sigma) supplemented with 10% FBS (Gibco, USA),
L-glutamine (2 mM, Jaber ibn Hayan, Iran), penicillin (100 IU/mL, Jaber ibn Hayan, Iran) and
streptomycin (100 µg/mL, Jaber ibn Hayan, Iran) under standard conditions and was sterilized by
0.22 µm microbiological filters (Millipore, Irland) after preparation and kept at 4°C before using.
The cells were subcultured in the ratio 1:3 twice per week. Passages 1–15 were used for
experiments. Cells were seeded at a density of 5000 cells/well in 96-well plates (Nunc,
Denmark). Incubations with various concentrations of the extracts were started 24 h after
seeding and continued for 24 and 48 hours.
Extraction Procedure
The shade dried aerial parts of each species (100 g) were chopped in small pieces and then
crushed into powder by a blinder. Each sample was macerated in ethanol 70% (v/v) for 48 hours
and then extracted by a percolator. The extracted solutions were concentrated at 50°C under
reduced pressure to dryness.
Cytotoxicity assay
The cytotoxic effect of obtained extracts against previously mentioned human tumor cell lines
was determined by a rapid colorimetric assay, using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide, Sigma, Germany] and compared with untreated controls (15).
This assay is based on the metabolic reduction of soluble MTT by mitochondrial enzyme activity
of viable tumor cells, into an insoluble colored formazan product, which can be measured
spectrophotometrically after dissolving in dimethyl sulfoxide (DMSO, Significancema,
Germany) (16). Briefly, 200 µL of HepG-2 cells (2.5
×
104 cells per ml mL of media) were
seeded in ten columns of 96-well microplates and incubated for 24 h (37°C, 5% CO2 air
humidified). Then, for 8 columns, 20 µL of prepared concentrations (200, 400, 600, 800, 1000,
1200, 1600 and 3200 µg/mL) of each extract was added to each column and incubated for
another 24 and 48 h in the same condition. Two columns of each microplate were specified to
blank (containing only DMEM) and control (containing 5% ethanol 96%), respectively. To
evaluate cell survival, 20 µL of MTT solution (5 mg/mL in phosphate buffer solution) was added
to each well and incubated for 3-4 h. Then, almost all old medium containing MTT was gently
replaced by 200 µL of DMSO and 20 µL of glycine buffer (0.1 M, Biogen, Iran) and then
pipetted to dissolve any formed formazan crystals. The first column contained blank (20 µL
glycine, 200 µL DMSO) and last column contained culture without cells. Finally, the
microplates were incubated at room temperature for 30 min. The same procedure was carried
out for Hep-2 cells as well.
The absorbance of each well was measured by an ELISA reader (STAT FAX 303, USA) at a
wavelength of 570 nm. Determination of percent of growth inhibition was carried out using the
following equilibrium: Cell survival (%) = T/C × 100
Where C is the mean absorbance of control group and T is the mean absorbance of test group.

Pharmacologyonline 3: 327-339 (2009) Emami et al.
330
Statistical analysis
The data were expressed as standard error of mean (SEM) of 8 independent experiments.
Wherever appropriate, the data were subjected to statistical analysis by one-way analysis of
variance (one-way ANOVA) followed by Tukey-Kramer test and Paired-Sample T Test for
multiple comparisons. A value of p<0.05 was considered significant. SPSS 11.5 software was
used for the statistical analysis. Curves were plotted by Graphpad Prism 4 Demo.
Results
24-hour exposure
Results of the MTT cytotoxicity assay for different concentrations of ethanol extracts of A.
kulbadica, A. diffusa, A. sieberi, A. santolina and A. turanica against HepG-2 and Hep-2 cell
lines are presented in Figures 1 through 5, respectively. Results showed a concentration-
dependent toxicity for all the extracts and the overall toxicity on HepG-2 cells is more than that
on Hep-2 cells. The calculated IC50 values for the above mentioned Artemisia species against
Hep2 cells were 951.11, 1054.75, 231.59, 1093.82 and 975.54 and against HepG2 cells were
<200, 520.70, <200, 514.82 and 259.49, respectively. Evaluation of IC50 indicated that the
extract of A. kulbadica was most toxic against HepG2 and A. sieberi was the most toxic against
Hep2 cells.
48-hour exposure
Results have shown a similar pattern to 24-hour exposure experiments with higher toxicity,
possibly due to longer exposure (results not shown).
The order of survival cells are as follows:
HepG2 (After 24 h):
A. diffusa > A. santolina > A. turanica > A. sieberi > A. kulbadica
Hep2 (After 24 h):
A. santolina > A. kulbadica = A. turanica = A. diffusa > A. sieberi
According to graphs, Minimum toxic dose of extracts of Artemisia as follows:
A. kulbadica=400 µg/ml A. sieberi=800 µg/ml
A. turanica=1000 µg/ml A. santolina=1600 µg/ ml A. diffusa=1600µg/ml

Pharmacologyonline 3: 327-339 (2009) Emami et al.
331
**
*** *** *** *** *** *** ***
Discussion
In our investigation, the in vitro toxicity of the ethanol extracts of some Iranian Artemisia
species were shown against two cancer cell lines: Hep-2 and HepG-2. This is a concentration-
dependent effect in the range of 200 through 3200 µg/mL. Collectively, this toxicity is stronger
against HepG-2 compared with Hep-2 cell lines.
The toxicity of Artemisia species on cancer cells has been shown in vitro (17-19) and in vivo (7,
20). Also, we previously have shown a similar effect for some other Iranian Artemisia species
(21). Artemisinin, the active ingredient of Artemisia annua, showed cell toxicity against human
lymphoid leukemia (Molt-4) cells (22). Cesquiterpenlactones, terpenoids and flavonoids are
other antitumor substances extracted from Artemisia species (23, 24). Artesunate, a
semisynthetic derivative of artemisinin, showed both in vitro and in vivo anticancer effects (25).
It also may induce antiangiogenic effects that may contribute to its anticancer effects. The
predominant effect of the extracts or the active ingredients of Artemisia species is apoptosis.
They induce apoptosis in various cell lines via activation of caspases, depolarization of the
mitochondrial membrane potential and down-regulation of Bcl-2 expression (18) or cell cycle
arrest (26, 27). Hu, et al. observed morphological changes typical of apoptosis, including
condensed chromatin and a reduction in volume with exposure of another human hepatoma cell
line to components of Artemisia capillaries thunberg (26). Kim et al. (2007) suggested the use
of Artemisia fukudo as a preventive measure against cancer (27). Toxicity of Artemisia species
on HepG2 observed through inhibition of transeaminase (23).
0
0(alc)
200
400
600
800
1000
1200
1600
3200
0
25
50
75
100
Concentration(µ /ml)
%Absorption
Concentration (µg/ml)
0(alcohol)
Toxicity of A. Kulbadica a
g
ainst

Pharmacologyonline 3: 327-339 (2009) Emami et al.
332
Figure 1- Results of the MTT assay of different concentrations of Artemisia kulbadica on
HepG2 and Hep2 cells.
The cell lines were exposed to the plant extract for 24 hours and the results of MTT
viability assay at 570 nm were shown as mean ± SEM (n=8 in each concentration).
p value < 0.001): ***و **: 0.001 < p value < 0.01و(*: 0.01 < p value < 0.05
0
0(alc)
200
400
600
800
1000
1200
1600
3200
0
25
50
75
100
conc(µ /ml)
%Abs
Concentration (µg/ml)
0(alcohol)
%Absorption
Toxicit
y
of A.
s
ieberi a
g
ainst He
p
2
Concentration (µg/ml)
%Absorption
**
**
**
*** ***
***
***
***
Toxicit
y
of A.
s
ieberi a
g
ainst He
p
-G2
0(alcohol)

Pharmacologyonline 3: 327-339 (2009) Emami et al.
333
0
0(alc)
200
400
600
800
1000
1200
1600
3200
0
25
50
75
100
conc(µ /ml)
%Abs
Figure 2- Results of the MTT assay of different concentrations of Artemisia sieberi on
HepG2 and Hep2 cells.
The cell lines were exposed to the plant extract for 24 hours and the results of MTT
viability assay at 570 nm were shown as mean ± SEM (n=8 in each concentration).
p value < 0.001): ***و (**: 0.001 < p value < 0.01
**
**
** ** ** **
***
**
%Absorption
Concentration (µg/ml)
0(alcohol)

Pharmacologyonline 3: 327-339 (2009) Emami et al.
334
0
0(alc)
200
400
600
800
1000
1200
1600
3200
0
25
50
75
100
conc(µ /ml)
%Abs
0
0(alc)
200
400
600
800
1000
1200
1600
3200
0
25
50
75
100
conc(µ /ml)
%Abs
Figure 3- Results of the MTT assay of different concentrations of Artemisia turanica on
HepG2 and Hep2 cells.
The cell lines were exposed to the plant extract for 24 hours and the results of MTT
viability assay at 570 nm were shown as mean ± SEM (n=8 in each concentration).
p value < 0.001): ***و **: 0.001 < p value < 0.01و(*: 0.01 < p value < 0.05
***
*
**
**
**
***
***
***
0(alcohol)
Concentration (µg/ml)
%Absorption
Toxicit
y
of A. turanica a
g
ainst
*
*
*
**
**
**
***
0
(
alcohol
)
Concentration
(/l)
%Absor
p
tion
*
Toxicit
y
of A. turanica a
g
ainst

Pharmacologyonline 3: 327-339 (2009) Emami et al.
335
0
0(alc)
200
400
600
800
1000
1200
1600
3200
0
25
50
75
100
conc(µ /ml)
%Abs
0
0(alc)
200
400
600
800
1000
1200
1600
3200
0
25
50
75
100
conc(µ /ml)
%Abs
Figure 4- Results of the MTT assay of different concentrations of Artemisia santolina on
HepG2 and Hep2 cells.
The cell lines were exposed to the plant extract for 24 hours and the results of MTT
viability assay at 570 nm were shown as mean ± SEM (n=8 in each concentration).
p value < 0.001): ***و **: 0.001 < p value < 0.01و(*: 0.01 < p value < 0.05
***
***
*
*
**
**
***
***
0
(
alcohol
)
Concentration
(/l)
%Absor
p
tion
Toxicit
y
of A. santolina a
g
ainst
*
*
*
*
**
**
**
***
0
(
alcohol
)
Concentration
(/l)
%Absor
p
tion
Toxicit
y
of A. santolina a
g
ainst

Pharmacologyonline 3: 327-339 (2009) Emami et al.
336
0
0(alc)
200
400
600
800
1000
1200
1600
3200
0
25
50
75
100
conc(µ /ml)
%Abs
0
0(alc)
200
400
600
800
1000
1200
1600
3200
0
10
20
30
40
50
60
70
80
90
100
110
conc(µ /ml)
%Abs
Figure 5- Results of the MTT assay of different concentrations of Artemisia diffusa on
HepG2 and Hep2 cells.
The cell lines were exposed to the plant extract for 24 hours and the results of MTT
viability assay at 570 nm were shown as mean ± SEM (n=8 in each concentration).
p value < 0.001): ***و **: 0.001 < p value < 0.01و(*: 0.01 < p value < 0.05
***
*
*
** **
**
***
***
0
(
alcohol
)
Concentration (µg/ml)
%Absor
p
tion
Toxicit
y
of A. diffusa a
g
ainst He
p
-
*
*
*
**
**
**
**
***
0
(
alcohol
)
Concentration
(/l)
%Absor
p
tion
Toxicit
y
of A. diffusa a
g
ainst He
p
2

Pharmacologyonline 3: 327-339 (2009) Emami et al.
337
Artemisinin itself produced rapid apoptosis rather than necrosis against human lymphoid
leukemia (Molt-4) cells (22). Based on these previous studies, the mechanism of toxicity of
Artemisia species is predominantly apoptosis, although, necrosis is also another possibility,
especially in higher concentrations.
In our experiments, the extracts showed more potent activity against HepG2 cells than Hep2
cells. HepG2 cells are of liver origin and express a wide range of phase I and phase II enzymes
such as cytochrome P450 enzymes, hydrolase, nitroreductase, catalase, peroxidase,
NAD(P)H:cytochrome c reductase, cytochrome P450 reductase, epoxide hydrolase,
sulfotransferase, glutathione S-transferase (GST), and N-acetyl transferase (28). Some of these
enzymes present in higher concentrations in growing than in confluent cells (29). Our
experiments were carried out in the growing phase of HepG2 and Hep2 cells, thus a high
metabolic activity could be estimated. Eupatilin, a pharmacologically active flavone derived
from Artemisia species, is extensively metabolized by cytochrome P450 (CYP) and UDP-
glucuronosyltransferase (UGT) enzymes in human liver microsomes. Activation reactions with
rifampin have been shown in HepG2 cells, a drug that undergo metabolic activation in the liver
and causes hepatotoxicity (30). In our previous study, similar results were observed in these two
cell lines (31). Because of high metabolic capacity of HepG2 cells for activation and
deactivation of xenobiotics, part of the higher toxicity of the plants extracts against HepG-2
compared with Hep2 cells may be due to the activation reactions of ingredients of the extracts.
Complementary studies will elucidate the uncovered aspects of anticancer toxicity of these
plants.
Acknowledgement
The authors would like to thank the authorities in research council of Mashhad University of
Medical Sciences and School of Pharmacy (MUMS) for their supports.
References
1- Mucciaralli M, Maffel M. Introduction of the genus In: Wright CW. ed. Artemisia,
Taylor and Francis, London, 2002: 1-50.
2- Polyakov P. Artemisia In: Shishkin BK, ed. Flora of the USSR (English translation),
vol. 29, Bisnen Singh Scientific Books, Koeringstein, Germany, 1995: 488-489.
3- Podlech D. Compositae VI Anthemideae In: Rechinger KH, ed. Flora Iranica, No. 158,
Graz, Akademische Druck-u, Verlagsansalt, 1986: 159-223.
4- Ghahreman A, Attar F. Biodiversity of plant species in Iran, vol. 1, Tehran University
Publication, Tehran, 1999: 41-42.
5- Emami SA, Aghazari F. Les Phanerogames endemiques de la flore d' Iran,
publication de l' Universite' d' Iran des Sciences Medicales, Téhéran, 2008: 349.
6- Mozaffarian V. Compositae: Anthemideae & Echinapeae. In: Assadi M, ed. Flora of
Iran No. 59, Research Institute of Forests and Rangelands 2008: 199-261 (In Persian)
7- Efferth T. Willmar Schwabe Award 2006: Antiplasmodial and Antitumor Activity of
Artemisinin - From Bench to Bedside, Planta Med 2007; 73: 299-309.
8- Allen PC, Danforth HD, Augustine PC. Dietary modulation of avian coccidiosis. Int J
Parasitol 1998; 28: 1131-1140.

Pharmacologyonline 3: 327-339 (2009) Emami et al.
338
9- Zheng GQ. Cytotoxic terpenoides and flavonoides from Artemisia annua. Planta Med
1994; 60: 54-57.
10- Farzaneh M, Ahmadzadeh M, Hadian J, Tehrani AS. Chemical composition and
antifungal activity of the essential oils of three species of Artemisia on some soil-borne
phytopathogens. Commun Agric Appl Biol Sci 2006; 71: 1327-1333.
11- Kordali S, Kotan R, Mavi A, Cakir A, Ala A, Yildirim A, Agric J. Determination of the
chemical composition and antioxidant activity of the essential oil of Artemisia
dracunculus and of the antifungal and antibacterial activities of Turkish Artemisia
absinthium, A. dracunculus, Artemisia santonicum, and Artemisia spicigera essential
oils. Food Chem 2005; 53: 9452-9458.
12- Kordali S, Cakir A, Mavi A, Kilic H, Yildirim A. Screening of chemical composition
and antifungal and antioxidant activities of the essential oils from three Turkish
Artemisia species. J Agric Food Chem 2005; 53: 1408-1416.
13- Szeto YT, Benzie IFF. Is the yin-yang nature of Chinese herbal medicine equivalent to
antioxidation-oxidation? J Ethnopharmacol 2006; 108: 361-366.
14- Bhatt LR, Lim JA, Chai KY, Kang JI, Oh HK, Baek SH. Antioxidant and antimicrobial
activities of essential oil from Artemisia vulgaris. Nat Prod Sci 2006; 12: 226-231.
15- Mosmann T (1983): Rapid colorimetric assay for cellular growth and survival:
Application to proliferation and cytotoxicity assays. J Immunol Methods 1983; 65: 55-
63.
16- Carmichale J, De Graff WG, Gazdar AF, Mina JD, Mitchell JB. Evaluation of a
tetrazolium based semiautomated colorimetric assay: Assessment of chemosensitivity
testing. Cancer Res 1987; 47: 936-942.
17- Willoughby Sr JA, Sundar SN, Cheung M, Tin AS, Modiano J, Firestone GL.
Artemisinin blocks prostate cancer growth and cell cycle progression by disrupting Sp1
interactions with the cyclin-dependent kinase-4 promoter and inhibiting CDK4 gene
expression. J Biol Chem 2009; 284:2203-13.
18- Sarath VJ, So CS, Young DW, Gollapudi S. Artemisia princeps var orientalis induces
apoptosis in human breast cancer MCF-7 cells. Anticancer Res 2007; 27:3891-3898.
19- Mercer AE, Maggs JL, Sun XM, Cohen GM, Chadwick J, O'Neill PM. Park BK.
Evidence for the involvement of carbon-centered radicals in the induction of apoptotic
cell death by artemisinin compounds. J Biol Chem 2007; 282:9372-9382.
20- Lai H, Singh NP. Oral artemisinin prevents and delays the development of 7,12-
dimethylbenz[a]anthracene (DMBA)-induced breast cancer in the rat. Cancer lett 2006;
231:43-8.
21- Tan RX, Zheng WF, Tang HQ. Biologically active substances from genus Artemisia.
Planta Med 1998; 64: 295-302.
22- Singh NP, Lai HC. Artemisinin induces apoptosis in human cancer cells. Anticancer
Res 2007; 24: 2277-2280.
23- Suffiness M, Pezzuto JM. Methods in plant biochemistry, Academic Press, Sandiego,
1991: 71-131.
24- Wang Q, Wu Lu, Li AY, Zhao Y, Wang NP. Experimental studies of antitumor effect
of artesunate on liver cancer. China Journal of Chinese Materia Medica 2001; 26: 707-
708, 720.
25- Li PC, Lam E, Roos WP, Zdzienicka MZ, Kaina B, Efferth T. Artesunate derived from
traditional Chinese medicine induces DNA damage and repair. Cancer Res 2008;
68:4347-4351.

Pharmacologyonline 3: 327-339 (2009) Emami et al.
339
26- Hu YQ, Tan RX, Chu MY, Zhou J. Apoptosis in human hepatoma cell line SMMC-
7721 induced by water-soluble macromolecular components of Artemisia capillaries
Thunberg. China Journal of Chinese material medica 2001; 26: 707-708, 720.
27- Kim KN, Lee JA, Yoon WJ, Kim JY, Song GP, Park SY. The cytotoxicity of Artemisia
fukudo extracts against HL-60 cells. Journal of the Korean Society of Food Science and
Nutrition 2007; 36: 819-824.
28- Knasmüller S, Parzefall W, Sanyal R, Ecker S, Schwab C, Uhl M, Mersch-Sundermann
V, Williamson G, Hietsch G, Langer T, Darroudi F, Natarajan AT. Use of
metabolically competent human hepatoma cells for the detection of mutagens and
antimutagens. Mutat Res 1998; 402: 185–202.
29- Alexandre E, David P, Viollon C, Wolf P, Jaeck D, Azimzadeh A, Nicod L, Boudjema
K, Richert L. Expression of cytochromes P-450 2E1, 3A4 and 1A1/1A2 in growing and
confluent human HepG2 hepatoma cells-effect of ethanol. Toxicol In Vitro 1999; 13:
427-435.
30- Vahdati-Mashhadian N, Jaafari MR, Nosrati A. Differential toxicity of rifampin on
HepG2 and Hep2 cells using MTT test and electron microscope. Pharmacologyonline
2007; 3: 405-413.
31- Vahdati-Mashhadian, N., Emami, S. A., Oghazian, M. B. and Vosooq, R. (2009). The
cytotoxicity evaluation of seven species of Artemisia on human tumor cell lines.
Pharmacologyonline 1: 229-242.