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A study of toxicity and differential gene expression in murine liver following exposure to anti-malarial drugs: amodiaquine and sulphadoxine-pyrimethamine

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  • 1Email author
Malaria Journal201110:109

  • Received: 6 July 2010
  • Accepted: 2 May 2011
  • Published:



Amodiaquine (AQ) along with sulphadoxine-pyrimethamine (SP) offers effective and cheaper treatment against chloroquine-resistant falciparum malaria in many parts of sub-Saharan Africa. Considering the previous history of hepatitis, agranulocytosis and neutrocytopenia associated with AQ monotherapy, it becomes imperative to study the toxicity of co-administration of AQ and SP. In this study, toxicity and resulting global differential gene expression was analyzed following exposure to these drugs in experimental Swiss mice.


The conventional markers of toxicity in serum, oxidative stress parameters in tissue homogenates, histology of liver and alterations in global transcriptomic expression were evaluated to study the toxic effects of AQ and SP in isolation and in combination.


The combination therapy of AQ and SP results in more pronounced hepatotoxicity as revealed by elevated level of serum ALT, AST with respect to their individual drug exposure regimen. Furthermore, alterations in the activity of major antioxidant enzymes (glutathione peroxidase, superoxide dismutase, catalase, glutathione reductase), indicating the development of oxidative stress, was more significant in AQ+SP combination therapy. cDNA microarray results too showed considerably more perturbed gene expression following combination therapy of AQ and SP as compared to their individual drug treatment. Moreover, a set of genes were identified whose expression pattern can be further investigated for identifying a good biomarker for potential anti-malarial hepatotoxicity.


These observations clearly indicate AQ+SP combination therapy is hepatotoxic in experimental Swiss mice. Microarray results provide a considerable number of potential biomarkers of anti-malarial drug toxicity. These findings hence will be useful for future drug toxicity studies, albeit implications of this study in clinical conditions need to be monitored with cautions.


  • Malaria
  • Amodiaquine
  • Murine Liver
  • Drug Induce Toxicity
  • Major Antioxidant Enzyme


Malaria remains to be the major killer disease in the developing countries that affects lives of more than 500 million people and kills about two million of them annually [1]. Most of the drugs that are used to treat malaria can be broadly grouped into 4-aminoquinolines, 8-aminoquinolines, anti-folates, artemisinin derivatives, hydroxyl naphthoquinones and certain class of antibiotics, such as doxycycline and clindamycin. 4-aminoquiniline derivatives, such as chloroquine and amodiaquine (AQ), have been the first-line drugs against malaria for past several decades. Development of resistance against these drugs in several parts of world necessitated the use of other drugs along with it for efficient treatment. Malaria treatment guidelines issued by WHO also recommends the use of AQ and SP combinations for the treatment of chloroquine-resistant malaria [24]. Many clinical trials and field studies, carried primarily in African countries, showed that AQ in combination with SP was very effective in controlling cases of malaria [57]. Although resistance against sulphadoxine-pyrimethamine has also been reported in parts of East Africa [810], it remains a good choice for rest of the world, including West Africa [11]. Notwithstanding their utility in controlling malaria, most of these anti-malarials are also associated with risk of drug-induced toxicity [1214]. In spite of wide use of AQ and SP as anti-malarials, there is dearth of scientific literature describing their potential toxicity [15].

Liver is a vital organ of body and mainly involved in drug metabolism and its biotransformation. Its unique position and crucial link with gastro-intestinal tract renders it highly vulnerable to drug induced toxicity [16, 17]. Previously, Noel et al. had described toxicity and gene expression alterations in murine liver following exposure to the anti-relapse anti-malarial drugs primaquine [18] and bulaquine [19]. High throughput gene expression profiling facilitates prediction of toxicity and interpretation of mechanism of toxicity based on distinct gene expression changes. The simplest approach to identify genes of potential interest through several related experiments is to search for those that are consistently either up- or down-regulated [1820]. Therefore, an attempt was made to delineate the mechanism of anti-malarial drug toxicity in liver tissue following exposure to AQ and SP combination in murine models.


Animal groups, drug administration and tissue collection

10-12 weeks old, male Swiss albino mice (Mus musculus), weighing 25-30 g (Central Drug Research Institute, Lucknow, India) were randomly assigned to control and treatment groups. All animal procedures were performed following IAEC approval (115/07/Toxicol./IAEC dated 11.9.2007) and in compliance to institutional animal ethics guidelines. The animals were acclimated to optimal conditions of temperature (25 ± 2°C) and light/dark cycle (12 h each) before initiation of drug administration. The doses for AQ and SP were calculated from human therapeutic doses [21] based on equivalent body surface area index [22]. The duration of dosing in mice was also similar to the human therapeutic regime. Animals were divided into four groups each consisting of six animals and were given following dosages orally.

Group 1: 1% DMSO-treated controls, for three consecutive days

Group 2: AQ, 120 mg/kg for three consecutive days

Group 3: 300 mg/kg sulphadoxine and 15 mg/kg pyrimethamine on day one

Group 4: 120 mg/kg AQ and SP, 300 mg/kg and 15 mg/kg respectively, on day one followed by 120 mg/kg AQ, on day two and three

All animals were sacrificed by cervical dislocation on day four of study and liver was taken out after perfusion with normal saline and a part of it is kept at -70°C until further analysis. Prior to sacrifice blood was taken out from cardiac puncture from each animal and left undisturbed for 30 minutes for serum separation. A part of liver tissue was immediately fixed in 10% formal saline for histological investigations.

Serum biochemistry and liver histology

Alanine aminotransferase (ALT), aspartate aminotransferase (AST) [markers of hepatotoxicity] levels were estimated in the serum with automated biochemical analyzer using the kits (Beckmann). Fixed liver tissues were washed overnight, dehydrated through graded alcohols and embedded in paraffin wax. Serial sections of 5 μm thickness were stained with haematoxylin and eosin (H&E) for histological examination.

Biochemical estimation of antioxidant enzymes in liver tissue fraction

Markers of oxidative stress {tissue levels of lipid peroxidation; LPO [a measure of malondialdehyde (MDA) concentration] and reduced glutathione level; GSH} and enzyme activities of major antioxidant enzymes (glutathione peroxidase, superoxide dismutase, catalase, glutathione reductase) were estimated in liver tissue homogenates using standard tests [2327].

RNA isolation, cDNA labeling and hybridization

50 mg frozen liver tissue was crushed in liquid nitrogen and immediately homogenized (Heidolph, Germany) in 1 ml of TRI reagent (Sigma, St. Louis, MO, USA) to isolate total RNA. RNA samples with approximately 2:1 ratio of 28S:18S rRNA and 260/280 values ≥ 1.8 were used for gene expression analysis. Equal amount of RNA from individuals of the same group was pooled to eliminate inter-individual variations. 25 μg of pooled RNA was converted into labeled cDNA using CyScribe First Strand cDNA-labeling kit (Amersham, Buckinghamshire, UK) following manufacturer's protocol. Labeled cDNA was purified with GFX columns as per manufacturer's guidelines and subsequently concentrated by evaporation under vacuum after estimating the percent incorporation of the dyes with a spectrophotometer (Thermo, Waltham, MA, USA). Dye swap technical replicate experiments were performed with aliquots of same RNA preparation to address inconsistencies regarding dye incorporation and other technical means of variance. The Cy5- and Cy3-labeled cDNA samples were mixed in CyScribe Hyb buffer (Amersham, Buckinghamshire, UK) containing 10 μg/ml sheared salmon sperm DNA and 10 μg/ml yeast tRNA (Ambion, Austin, Texas, USA) as blocking agents. The labeled sample was hybridized to mouse 22.4k arrays [28] for 18 h at 42°C.

Scanning and microarray data analysis

The arrays were washed and subsequently scanned to collect raw data with Array Scanner III supported with Image-Quant version 5 (Molecular Dynamics). Intensity values were extracted from the scanned images with ArrayVision version 8 (Imaging research, GE healthcare Biosciences Corp., Piscataway, NJ, USA). Raw intensity data was analyzed with Avadis Express version 4.3 (Strand life Sciences, Bangalore, India) and the background corrected intensities were LOWESS normalized (Cy5 against Cy3) to obtain log (base 2) ratios. Furthermore, log2 values of duplicate spots were averaged in order to get a single mean value to perform k-means clustering with MeV version 3.1 [TM4, The Institute of Genomic Research [29]]. Each expression cluster was further clustered hierarchically with Euclidean distance matrix and average linkage to identify gene with similar expression patterns. Raw and log transformed data (series accession no. GSE 17392) has been submitted to Gene Expression Omnibus database [30] and conforms to MIAME guidelines developed by microarray gene expression data (MGED) society.

Real time-PCR

mRNA was reverse transcribed according to the manufacturer's instruction (First Strand cDNA Synthesis Kit for RT-PCR, Invitrogen, California, USA). PCRs were performed on a Light Cycler 480 System (Roche Diagnostics) in 96-well plates. Each reaction was carried out in 20 μl reaction volume comprising of SYBR Green qPCR Master Mix (Invitrogen, California, USA), cDNA template, 200 nM of forward and reverse primers and nuclease-free water. Serial dilutions of genomic DNA (250-0.08 ng) were used to generate a quantitative PCR standard curve. The LightCycler protocol was: 2 min. of UDG incubation (Invitrogen, California USA) at 50°C followed by 10 min. of 95°C hot-start enzyme activation; 40 cycles of 95°C denaturation for 15 s, 60°C annealing and elongation. Melting curve analysis temperatures were 95°C for 5 s, 70°C for 60 s, and then heating to 95°C. Water was used as the template for negative control amplifications included with each PCR. Data were analyzed using the Roche LightCycler 480 software and Cp was calculated by the Second Derivate Maximum Method [31]. The amount of the target mRNA was examined and normalized to the GAPDH gene mRNA. The relative expression ratio of a target gene was calculated as described by Pfaffl [32], based on real-time PCR efficiencies. Results reported were obtained from at least three biological replicates and PCR runs were repeated at least twice.

Statistical analysis

Data were expressed as the mean ± standard error of the means (S.E.M.). Group means were compared by one-way analysis of variance (ANOVA) with Newman-Keuls post analysis test. The differences in the data obtained were considered statistically significant when the P-value was less than 0.05. All statistical analysis was done through using Prism ver.5 (GraphPad Software Inc., USA).


Effect of amodiaquine and sulphadoxine-pyrimethamine treatment on the biomarkers of hepatotoxicity and oxidative stress

Treatment of AQ at 120 mg/kg does not impart hepatotoxicity or oxidative stress, as levels of ALT, AST, LPO and GSH were comparable to that of untreated control. Although administration of SP does not cause any elevation in level of ALT or AST, it causes appreciable oxidative stress, as a significant elevation in LPO and a decrease in GSH were observed in mice dosed with SP. Interestingly, co-administration of AQ and SP (i.e. AQ+SP) causes both hepatotoxicity as well as oxidative stress as evident from marked increase in ALT, AST, LPO and decrease in GSH (Figure 1a, b, c and 1d).
Figure 1
Figure 1

(a - h) Assessment of markers of hepatotoxicity and oxidative stress following exposure of AQ, SP and AQ+SP. Group 1: untreated control; Group 2: treated with AQ, 120 mg/kg body wt[AQ]; Group 3: treated with sulphadoxine (300 mg/kg) and pyrimethamine (15 mg/kg) [SP]; Group 4: Co-treatment of AQ and SP[AQ+SP]; [*(P < 0.05), ** (P < 0.01), *** (P < 0.001)].

Effect of amodiaquine and sulphadoxine-pyrimethamine treatment on antioxidant enzymes in liver tissue fraction

Effects of AQ and SP treatment on enzymatic activities of SOD, catalase, GR and GPx, which are the major antioxidant enzymes in liver tissue fraction, were investigated. SOD activity was not altered after AQ and SP treatment, while catalase and GPx activities were drastically reduced by the treatment of SP and AQ+SP. However, AQ administration did cause a moderate, statistically non-significant, increase in the activity of SOD and catalase. However, activity of GR was increased by administration of AQ and AQ+SP combination (Figure 1e, f, g and 1h).

cDNA Microarray analysis of differential gene expression in murine liver and kidney exposed to anti-malarials amodiaquine and sulphadoxine-pyrimethamine

Following AQ administration in murine liver, a total of 133 probes were differentially regulated, of which 60 were up-regulated and 73 down-regulated. Some of these are listed in Table 1. Major important up-regulated probes following AQ dosing included the TAP binding gene involved in antigen processing, the neogenin gene involved in ATP binding, the dihydropyrimidinase like 5 gene involved in axon guidance, the ankyrin repeat domain 6 gene involved in DNA binding and genes for GATA binding protein 2 involved in DNA binding and transcription. Some of the important down-regulated probes following AQ administration included the DEAD box polypeptide 6 gene involved in ATP-dependent helicase activity, the voltage dependent calcium channel L type alpha 1 C subunit gene involved in calcium channel activity, the lipoma HMGIC fusion partner-like 2 gene involved in general metabolism, and the GCN5 gene involved in N-acetyl transferase activity.
Table 1

List of important differentially expressed probes after administration of AQ in murine Liver.

Spot labels

Fold Change

Gene Name/Description

GO : Biological function



TAP binding protein

antigen processing



RAB39B, member RAS oncogene family

GTP binding




ATP binding



ATPase, Ca++ transporting, plasma membrane 1

ATP binding



Dihydropyrimidinase-like 5

axon guidance



Calcium channel, voltage-dependent, L type, alpha 1D subunit

calcium channel activity



Ankyrin repeat domain 6

DNA binding



GATA binding protein 2

DNA binding



Parathyroid hormone receptor 1

G-protein coupled receptor activity



Transmembrane protein with EGF-like and two follistatin-like domains 1

growth factor activity



Solute carrier family 38, member 1

L-glutamine transport



DNA segment, Chr 5, Wayne State University 178, expressed

phospholipid biosynthesis



DEAD (Asp-Glu-Ala-Asp) box polypeptide 6

ATP-dependent helicase activity



Calcium channel, voltage-dependent, L type, alpha 1C subunit

calcium channel activity



Nucleoporin 153

DNA binding



Suppressor of variegation 4-20 homolog 1 (Drosophila)

histone lysine N-methyltransferase activity



Lipoma HMGIC fusion partner-like 2




GCN5 general control of amino acid synthesis-like 2 (yeast)

N-acetyltransferase activity



Nuclear receptor subfamily 3, group C, member 2

transcription factor activity

Fold change (FC) >2(Up-regulated) and FC<-2 (Down-regulated) and P < 0.01.

Administration of SP in murine liver leads to differential regulation of 156 probes of which 90 were up-regulated and 66 down-regulated, some of which are listed in Table 2. Some of the important up-regulated probes following SP treatment included the DEAH box polypeptide 15 gene involved in ATP-dependent helicase activity, the transketolase gene involved in calcium ion binding, the procollagen type VI alpha 2 genes mainly involved in cell adhesion, the procollagen lysine 2-oxoglutarate 5-dioxygenase 2 genes involved in endopeptidase inhibitor activity and a gene coding for RNA binding motif protein X. Major down-regulated probes following SP administration included the CDC42 effector protein 1 (Rho GTPase binding) involved in signal transduction, the serine (or cysteine) peptidase inhibitor clade B member 6a gene involved in endopeptidase inhibitor activity and the cytochrome c oxidase subunit VIb polypeptide 2 gene involved in electron transfer.
Table 2

List of Important differentially expressed probes after administration of SP in murine Liver.

Spot labels

Fold Change

Gene Name/Description

GO : Biological function



Histone deacetylase 9

Histone deacetylase activity



DEAD (Asp-Glu-Ala-Asp) box polypeptide 42

ATP binding



DEAH (Asp-Glu-Ala-His) box polypeptide 15

ATP-dependent helicase activity




Calcium ion binding



Procollagen, type VI, alpha 2

Cell adhesion



Procollagen lysine, 2-oxoglutarate 5-dioxygenase 2

Endopeptidase inhibitor activity



Lymphocyte antigen 6 complex, locus G6C

Extracellular space



RNA binding motif protein, X chromosome retrogene

RNA binding



RNA binding motif protein 28

RNA binding



Eukaryotic translation initiation factor 1

Translation factor activity



CDC42 effector protein (Rho GTPase binding) 1

Signal transduction



Serine (or cysteine) peptidase inhibitor, clade B, member 6a

Endopeptidase inhibitor activity



Cytochrome c oxidase subunit VIb polypeptide 2




Tumor protein D52








RIKEN cDNA 4930471M23 gene




TBC1 domain family, member 19


Fold change (FC) >2(Up regulated) and FC<-2 (Down regulated) and P < 0.01.

Co-administration of AQ and SP for three consecutive days resulted in differential regulation of 231 probes, including 118 up-regulated and 113 down-regulated probes (Table 3). Major up-regulated probes following co-exposure of AQ and SP included genes having a cysteine and histidine-rich domain (CHORD) containing zinc-binding protein 1, mainly involved in calcium ion binding, the solute carrier family 28 (sodium-coupled nucleoside transporter) member 3 gene involved in ion transport, the Kelch-like 2 Mayven (Drosophila) gene involved in actin binding, the integrin beta 8 gene involved in cell adhesion, the gene for suppression of tumorigenicity (colon carcinoma) involved in cell migration and mortality factor 4 like 1 gene involved in cell proliferation. Some of the major down-regulated probes following AQ+SP treatment were the lysophospholipase 3 gene involved in acyltransferase activity, the mitogen-activated protein kinase 14 gene involved in ATP binding, the transforming growth factor beta receptor I gene involved in ATP binding, the procollagen type VI alpha3 gene involved in cell adhesion, the gene for microfibrillar-associated protein 4 involved mainly in cell adhesion, the BTB and CNC homology 2 genes involved in DNA binding and the CXXC finger 1 (PHD domain) gene also involved in DNA binding.
Table 3

List of Important differentially expressed probes after administration of AQ+SP in murine Liver.

Spot labels



Gene Name/Description

GO: Biological Function



Cysteine and histidine-rich domain (CHORD)-containing, zinc-binding protein 1

Calcium ion binding



Solute carrier family 28 (sodium-coupled nucleoside transporter), member 3

Integral to plasma membrane



Kelch-like 2, Mayven (Drosophila)

Actin binding



Integrin beta 8

Cell adhesion



Suppression of tumorigenicity 14 (colon carcinoma)

Cell migration



Mortality factor 4 like 1

Cell proliferation



Zinc finger, SWIM domain containing 4

Cellular component



Metal response element binding transcription factor 1

DNA binding



Regulatory factor X, 3 (influences HLA class II expression)

DNA binding



Polymerase (RNA) III (DNA directed) polypeptide F

DNA binding



Cytochrome b5 type B

Electron transport



GTP binding protein (gene overexpressed in skeletal muscle)

GTP binding



DNA segment, Chr 5, Wayne State University 178, expressed

Integral to membrane



DNA segment, Chr 18, ERATO Doi 653, expressed

Integral to membrane



Dystrobrevin binding protein 1

Muscle development



Dolichyl-di-phosphooligosaccharide-protein glycotransferase

N-linked glycosylation via asparagine



Adenosine deaminase, RNA-specific, B2

RNA binding



Solute carrier family 25 (mitochondrial carrier, glutamate), member 22

Transporter activity



Ubiquitin-conjugating enzyme E2D 2

Ubiquitin-dependent protein catabolism



Lysophospholipase 3

Acyltransferase activity



Mitogen activated protein kinase 14

ATP binding



Transforming growth factor, beta receptor I

ATP binding



Procollagen, type VI, alpha 3

Cell adhesion



Microfibrillar-associated protein 4

Cell adhesion



BTB and CNC homology 2

DNA binding



CXXC finger 1 (PHD domain)

DNA binding



GLI-Kruppel family member GLI3

DNA binding



Protein disulfide isomerase associated 6

DNA binding



AT rich interactive domain 5B (Mrf1 like)

DNA binding



Cytochrome b5 type B

Electron transport



Proteasome (prosome, macropain) subunit, beta type 2

Endopeptidase activity



Phosphatidylinositol 3-kinase, C2 domain containing, alpha polypeptide

Glycerophospholipid metabolism



RAS related protein 1b

GTP binding



Zinc metallopeptidase, STE24 homolog (S. cerevisiae)

Hydrolase activity



SH2-B PH domain containing signaling mediator 1

Intracellular signaling cascade



Malate dehydrogenase 2, NAD (mitochondrial)

Malate dehydrogenase activity



Ring finger protein (C3HC4 type) 19

Protein ubiquitination



Cleavage and polyadenylation specific factor 4

RNA binding

Fold change (FC) >2(Up regulated) and FC<-2 (Down regulated) and P < 0.01.

Real time quantitative PCR analysis showed that most of genes that are differentially expressed in microarray produced similar results in PCR too, i.e. the genes which are up-regulated in microarray are up-regulated in real time PCR too and vice versa (Table 4).
Table 4

List of genes with their description and expression results by Q-PCR and microarray following treatment with AQ and SP in murine liver.

Gene Symbol

Gene Name/Description

Q-PCR fold change

Microarray Result Up regulated(▲)/Down regulated ()


Adrenergic receptor, alpha 1b



Cytochrome P450, family 1, subfamily a, polypeptide 2



Cytochrome P450, family 2, subfamily e, polypeptide 1



Sterol-C4-methyl oxidase-like



Histocompatibility 2, class II,



RAS-related C3 botulinum



Minichromosome maintenance deficient 4 homolog



Vitamin K epoxide reductase complex,



Sterol-C5-desaturase (fungal ERG3, delta-5-desaturase) homolog



Alcohol dehydrogenase 1 (class I)



Growth arrest and DNA-damage-inducible 45 gamma



UDP glucuronosyltransferase 2 family, polypeptide B1



Minichromosome maintenance deficient 5 homolog



Glucagon receptor


Note the direction similarity among Q-PCR and microarray findings for gene expression results.


Amodiaquine and sulphadoxine-pyrimethamine offer a great potential as effective anti-malarial against chloroquine-resistant malaria and has been used in many parts of Africa as first-line anti-malarial treatment. However, considering the previous history of drug-induced hepatitis, oxidative stress associated with these drugs particularly AQ, it becomes imperative to study the toxicity associated with these drugs and their combination in liver tissue.

Dosages and duration of AQ and SP treatment in Swiss mice was according to the human therapeutic equivalent dose and malaria treatment regimen suggested by WHO guidelines [4]. This observation was that only co-treatment of AQ and SP (AQ+SP) as recommended combination therapy regimen produces toxicity and not their individual exposure. However, treatment with SP alone does produces appreciable oxidative stress leading to a conclusion that observed hepatotoxicity and oxidative stress in AQ+SP group might be a result of either SP toxicity alone or an additive effect of both these drugs. Interestingly, none of these drugs or drug combinations results in alterations in normal liver histology as no histopathological damage was observed in any sections of liver tissues (Figure 2).
Figure 2
Figure 2

Murine liver cross-sections treated with amodiaquine and sulphadoxine: (a) untreated control, (b) treated with 120 mg/kg of AQ, (c) treated with 300 mg/kg of sulphadoxine and 15 mg/kg of pyrimethamine (SP), (d) Co-exposure of 120 mg/kg AQ and 300 mg/kg sulphadoxine along with 15 mg/kg of pyrimethamine (AQ+SP).

Previous reports showed that anti-malarials, particularly chloroquine, produce oxidative stress in liver tissue [14], and it was also interesting to study the alteration in antioxidant profile of major antioxidant enzyme present in liver tissue fraction. Results showed that the activity of SOD was not affected either by the treatment of AQ or SP or their combination (AQ+SP). However, the level of GPx was significantly reduced in all three treatment groups and catalase activity was reduced in SP and AQ+SP group in murine liver fraction. The decrease in the activity of GPx observed in this study might be the result of a decrease in GSH content; a measure substrate in GPX catalyzed reaction. Interestingly, GR activity was observed to increase in AQ and AQ+SP. The alterations in activities of antioxidant enzymes of liver observed in the present study were an indication of oxidative injury brought by the AQ and SP dosing.

High throughput expression profiling facilitates the prediction and mechanism of toxicity based on distinct gene expression changes. Therefore, the study of differential gene expression in murine liver at high statistical stringency (i.e. P < 0.01 and expression fold change >2) clearly indicated that the molecular mechanism of AQ and SP induced oxidative stress. Furthermore, validation of microarray findings using qRT-PCR further substantiates these results, which is the most sensitive and accurate method for validating microarray-based differential expression of genes [33]. The pattern of differential expression of genes in combination therapy, i.e. the AQ+SP treated groups, were on an expected line with biochemical observations, showing more robust expression pattern than either of the drug given alone. Here the number of differentially expressed probes was 231, far more differentially expressed genes than AQ (133) or SP (156) alone. Of the 231 differentially expressed genes in murine liver after AQ+SP treatment, the number of up-regulated (118) and of down-regulated (113) probes was almost similar (Figure 3).
Figure 3
Figure 3

Total number of differentially expressed genes following exposure to anti-malarial drugs in murine liver.

GenMAPP and MAPPFinder tools [34] were utilized to enlist the various biological pathways that are perturbed following exposure to AQ, SP or their combination (AQ+SP). The pathways that are most affected are signaling pathways, carbohydrate metabolism, oxidative stress and drug metabolism (Figure 4). These observations suggest that anti-malarial drug exposure imparts stress in liver tissue causing changes in mRNA expression level of antioxidant pathway and major drug metabolism pathway.
Figure 4
Figure 4

Important biological pathways regulated by administration of all the three dose categories (AQ, SP and AQ+SP) in murine liver.

One of the many genes that are up-regulated in murine liver following exposure to AQ, SP and their co-treatment i.e. AQ+SP includes EPRS (glutamyl-prolyl tRNA synthetase). EPRS is a multifunctional aminoacyl-tRNA synthetase that catalyzes the aminoacylation of glutamic acid and proline tRNA species [35]. Sampath et al.[36] showed that EPRS has a regulated, noncanonical activity that blocks synthesis of ceruloplasmin. Fall in the level of ceruloplasmin which is the major copper carrier protein, is an indication of hepatic stress [37], so the elevation in the level of EPRS following anti-malarial drug treatment can explain the observed hepatic stress. Supervilin (SVIL) is another gene that is consistently up-regulated in murine liver following exposure to AQ, SP and their combination. This gene codes for a protein, which is tightly associated with both actin filaments and plasma membranes, suggesting that it forms a link between the actin cytoskeleton and the membrane. An up-regulated SVIL (which is required for membrane integrity) following drug treatment may be an explanation for the rise in lipid peroxidation level observed in the present study. It appears that membrane damage following anti-malarial drug treatment is an inducing factor for up regulation of supervillin. Some of the many genes that were up-regulated in the present study include HSP90ab1, PAWR, and IKbRb among others. An up-regulated HSP90ab1 indicates that anti-malarial drug exposure has resulted in the development of hepatic stress. The PAWR genes are found to be transcriptionally induced by apoptotic signals in the rat ventral prostate [38]. Woronicz et al.[39] observed that IKbRb activates NF-kappa-B when overexpressed and phosphorylate serine residues 32 and 36 of I-kappa-B-alpha and 19 and 23 of I-kappa-B-beta. Therefore, upregulated PAWR and IKbRb in murine liver is an indication of cellular toxicity and inflammatory responses within liver hepatocytes following anti-malarial exposure.

One of the several genes that were down-regulated following anti-malarial exposure in murine liver is the myotubularin related protein 2 (MTMR2) gene. The MTMR2 gene encodes a protein that belongs to the myotubularin family, which is characterized by the presence of a phosphatase domain. Berger et al.[40] determined that mouse MTMR2 gene dephosphorylates phosphatidylinositol 3-phosphate (PI3P) and phosphatidylinositol 3, 5-bisphosphate (PI3, 5P2) with high efficiency and peak activity at neutral pH. A perturbation in phosphatidylinositol pathway resulting from down regulated MTMR2 expression is an indication of disturbances of signaling pathways following anti-malarial treatment.


Both biochemical and microarray results suggest that combination therapy of AQ and SP are more damaging than their individual monotherapies. Microarray results further suggests that present anti-malarial combination therapies lead to inflammatory responses and perturbed signaling cascade leading to general hepatic stress as observed in biochemical evaluation of liver tissue. Furthermore, expression level of EPRS, SVIL, PAWR, and MTMR2 can be good markers for anti-malarial drug induced hepatotoxicity. Hence, the present study can help in understanding anti-malarial drug induced toxicity. However, the clinical implication of the study needs to be evaluated further with caution as this study in experimental mice may not hold equally good in case of malaria prophylaxis and treatment for human population.



This work was supported by grant to SKR from the Council of Scientific and Industrial Research (CSIR) network project (NWP0034). Authors SKM and PS are recipients of Senior Research Fellowship from Indian Council of Medical Research, and Department of Biotechnology, India respectively. The paper bears CDRI communication number 8051.

Authors’ Affiliations

Genotoxicity Laboratory, Toxicology Division, Central Drug Research Institute, CSIR, Lucknow, PIN 226 001, India


  1. Snow RW, Guerra CA, Noor AM, Myint HY, Hay SI: The global distribution of clinical episodes of Plasmodium falciparum malaria. Nature. 2005, 434: 214-217. 10.1038/nature03342.PubMed CentralView ArticlePubMedGoogle Scholar
  2. Sowunmi A: A randomized comparison of chloroquine, amodiaquine and their combination with pyrimethamine-sulfadoxine in the treatment of acute, uncomplicated, Plasmodium falciparum malaria in children. Ann Trop Med Parasitol. 2002, 96: 227-238. 10.1179/000349802125000763.View ArticlePubMedGoogle Scholar
  3. Lederman ER, Maguire JD, Sumawinata IW, Chand K, Elyazar I, Estiana L, Sismadi P, Bangs MJ, Baird JK: Combined chloroquine, sulfadoxine/pyrimethamine and primaquine against Plasmodium falciparum in Central Java, Indonesia. Malar J. 2006, 5: 108-10.1186/1475-2875-5-108.PubMed CentralView ArticlePubMedGoogle Scholar
  4. Malaria treatment guidelines: World Health Organisation (WHO), Geneva. 2010Google Scholar
  5. Bell DJ, Nyirongo SK, Mukaka M, Zijlstra EE, Plowe CV, Molyneux ME, Ward SA, Winstanley PA: Sulfadoxine-pyrimethamine-based combinations for malaria: a randomised blinded trial to compare efficacy, safety and selection of resistance in Malawi. PLoS One. 2008, 3: e1578-10.1371/journal.pone.0001578.PubMed CentralView ArticlePubMedGoogle Scholar
  6. Perez MA, Cortes LJ, Guerra AP, Knudson A, Usta C, Nicholls RS: Efficacy of the amodiaquine+sulfadoxine-pyrimethamine combination and of chloroquine for the treatment of malaria in Cordoba, Colombia, 2006. Biomedica. 2008, 28: 148-159.View ArticlePubMedGoogle Scholar
  7. Sowunmi A, Balogun T, Gbotosho GO, Happi CT, Adedeji AA, Bolaji OM, Fehintola FA, Folarin OA: Activities of artemether-lumefantrine and amodiaquine-sulfalene-pyrimethamine against sexual-stage parasites in falciparum malaria in children. Chemotherapy. 2008, 54: 201-208. 10.1159/000140463.View ArticlePubMedGoogle Scholar
  8. The East African Network for Monitoring Antimalarial Treatment (EANMAT): The efficacy of antimalarial monotherapies, sulphadoxine-pyrimethamine and amodiaquine in East Africa: implications for sub-regional policy. Trop Med Int Health. 2003, 10: 860-867.View ArticleGoogle Scholar
  9. Talisuna AO, Nalunkuma-Kazibwe A, Bakyaita N, Langi P, Mutabingwa TK, Watkins WW, Van Marck E, D'Alessandro U, Egwang TG: Efficacy of sulphadoxine-pyrimethamine alone or combined with amodiaquine or chloroquine for the treatment of uncomplicated falciparum malaria in Ugandan children. Trop Med Int Health. 2004, 9: 222-229. 10.1046/j.1365-3156.2003.01187.x.View ArticlePubMedGoogle Scholar
  10. Discussion Paper for the RBM PARTNERS' MEETING. Edited by: Salle B. 2002, WHO, Geneva, 26-28. []
  11. White NJ: Antimalarial drug resistance. J Clin Invest. 2004, 113: 1084-1092.PubMed CentralView ArticlePubMedGoogle Scholar
  12. Dzur JR: Letter: Quinidine hepatotoxicity. JAMA. 1976, 235: 908-View ArticlePubMedGoogle Scholar
  13. Farver DK, Lavin MN: Quinine-induced hepatotoxicity. Ann Pharmacother. 1999, 33: 32-34. 10.1345/aph.18172.View ArticlePubMedGoogle Scholar
  14. Pari L, Murugavel P: Protective effect of alpha-lipoic acid against chloroquine-induced hepatotoxicity in rats. J Appl Toxicol. 2004, 24: 21-26. 10.1002/jat.940.View ArticlePubMedGoogle Scholar
  15. Taylor RJ, White NJ: Antimalarial drug toxicity. Drug Safety. 2004, 27: 25-61.View ArticlePubMedGoogle Scholar
  16. Lee WM: Drug-induced hepatotoxicity. N Engl J Med. 2003, 349: 474-485. 10.1056/NEJMra021844.View ArticlePubMedGoogle Scholar
  17. Minami K, Saito T, Narahara M, Tomita H, Kato H, Sugiyama H, Katoh M, Nakajima M, Yokoi T: Relationship between hepatic gene expression profiles and hepatotoxicity in five typical hepatotoxicant-administered rats. Toxicol Sci. 2005, 87: 296-305. 10.1093/toxsci/kfi235.View ArticlePubMedGoogle Scholar
  18. Noel S, Sharma S, Shanker R, Rath SK: Primaquine-induced differential gene expression analysis in mice liver using DNA microarrays. Toxicology. 2007, 239: 96-107. 10.1016/j.tox.2007.06.098.View ArticlePubMedGoogle Scholar
  19. Noel S, Sharma S, Shankar R, Rath SK: Identification of differentially expressed genes after acute exposure to bulaquine (CDRI 80/53) in mice liver. Basic Clin Pharmacol Toxicol. 2008, 103: 522-529. 10.1111/j.1742-7843.2008.00279.x.View ArticlePubMedGoogle Scholar
  20. Blomme EA, Yang Y, Waring JF: Use of toxicogenomics to understand mechanisms of drug-induced hepatotoxicity during drug discovery and development. Toxicol Lett. 2009, 186: 22-31. 10.1016/j.toxlet.2008.09.017.View ArticlePubMedGoogle Scholar
  21. WHO Malaria Factsheet. []
  22. Freireich EJ, Gehan EA, Rall DP, Schmidt LH, Skipper HE: Quantitative comparison of toxicity of anticancer agents in mouse, rat, hamster, dog, monkey, and man. Cancer Chemother Rep. 1966, 50: 219-244.PubMedGoogle Scholar
  23. Ohkawa H, Ohishi N, Yagi K: Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem. 1979, 95: 351-358. 10.1016/0003-2697(79)90738-3.View ArticlePubMedGoogle Scholar
  24. ELLMAN GL: Tissue sulfhydryl groups. Arch Biochem Biophys. 1959, 82: 70-77. 10.1016/0003-9861(59)90090-6.View ArticlePubMedGoogle Scholar
  25. Kakkar P, Das B, Viswanathan PN: A modified spectrophotometric assay of superoxide dismutase. Indian J Biochem Biophys. 1984, 21: 130-132.PubMedGoogle Scholar
  26. Sinha AK: Colorimetric assay of catalase. Anal Biochem. 1972, 47: 389-394. 10.1016/0003-2697(72)90132-7.View ArticlePubMedGoogle Scholar
  27. Wendel A: Glutathione peroxidase. Enzymatic Basis of Detoxication. Edited by: Jakoby WB. 1980, Academic Press, New York, 333-348.Google Scholar
  28. Website describing the distribution of probes in microarray slides used in the experiments. []
  29. Saeed AI, Sharov V, White J, Li J, Liang W, Bhagabati N: TM4: a free, open-source system for microarray data management and analysis. Biotechniques. 2003, 34: 374-378.PubMedGoogle Scholar
  30. Gene Expression Omnibus Database. []
  31. Luu-The V, Paquet N, Calvo E, Cumps J: Improved real-time RT-PCR method for high-throughput measurements using second derivative calculation and double correction. Biotechniques. 2005, 38: 287-293. 10.2144/05382RR05.View ArticlePubMedGoogle Scholar
  32. Pfaffl MW: A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001, 29: e45-10.1093/nar/29.9.e45.PubMed CentralView ArticlePubMedGoogle Scholar
  33. Chuaqui RF, Bonner RF, Best CJ, Gillespie JW, Flaig MJ, Hewitt SM, Phillips JL, Krizman DB, Tangrea MA, Ahram M, Linehan WM, Knezevic V, Emmert-Buck MR: Post-analysis follow-up and validation of microarray experiments. Nat Genet. 2002, 32 (Suppl): 509-514.View ArticlePubMedGoogle Scholar
  34. GenMAPP-Pathway analysis tool. []
  35. Cerini C, Kerjan P, Astier M, Gratecos D, Mirande M, Semeriva M: A component of the multisynthetase complex is a multifunctional aminoacyl-tRNA synthetase. EMBO J. 1991, 10: 4267-4277.PubMed CentralPubMedGoogle Scholar
  36. Sampath P, Mazumder B, Seshadri V, Gerber CA, Chavatte L, Kinter M, Ting SM, Dignam JD, Kim S, Driscoll DM, Fox PL: Noncanonical function of glutamyl-prolyl-tRNA synthetase: gene-specific silencing of translation. Cell. 2004, 119: 195-208. 10.1016/j.cell.2004.09.030.View ArticlePubMedGoogle Scholar
  37. Scheinberg IH, Gitlin D: Deficiency of ceruloplasmin in patients with hepatolenticular degeneration (Wilson's disease). Science. 1952, 116: 484-485. 10.1126/science.116.3018.484.View ArticlePubMedGoogle Scholar
  38. Johnstone RW, See RH, Sells SF, Wang J, Muthukkumar S, Englert C, Haber DA, Licht JD, Sugrue SP, Roberts T, Rangnekar VM, Shi Y: A novel repressor, par-4, modulates transcription and growth suppression functions of the Wilms' tumor suppressor WT1. Mol Cell Biol. 1996, 16: 6945-6956.PubMed CentralView ArticlePubMedGoogle Scholar
  39. Woronicz JD, Gao X, Cao Z, Rothe M, Goeddel DV: IkappaB kinase-beta: NF-kappaB activation and complex formation with IkappaB kinase-alpha and NIK. Science. 1997, 278: 866-869. 10.1126/science.278.5339.866.View ArticlePubMedGoogle Scholar
  40. Berger P, Bonneick S, Willi S, Wymann M, Suter U: Loss of phosphatase activity in myotubularin-related protein 2 is associated with Charcot-Marie-Tooth disease type 4B1. Hum Mol Genet. 2002, 11: 1569-1579. 10.1093/hmg/11.13.1569.View ArticlePubMedGoogle Scholar


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