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Treatment of pregnant BALB/c mice with sulphadoxine pyrimethamine or chloroquine abrogates Plasmodium berghei induced placental pathology Lalita Sharma, Geeta Shukla ⁎

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1383-5769/$ – see front matter © 2013 Published by Elsevier Ireland Ltd. http://dx.doi.org/10.1016/j.parint.2013.08.016

Please cite this article as: Sharma L, Shukla G, Treatment of pregnant BALB/c mice with sulphadoxine pyrimethamine or chloroquine abrogates Plasmodium berghei induced placental pathology, Parasitology International (2013), http://dx.doi.org/10.1016/j.parint.2013.08.016

PARINT-01146; No of Pages 1 Parasitology International xxx (2013) xxx



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Treatment of pregnant BALB/c mice with sulphadoxine pyrimethamine or chloroquine abrogates Plasmodium berghei induced placental pathology

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Lalita Sharma, Geeta Shukla ⁎

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Department of Microbiology, Panjab University, Chandigarh 160014, India

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• Treatment with antimalarials reduced parasitaemia in pregnant P. berghei infected mice. • Malondialdehyde levels and apoptotic cells were reduced in the placenta of treated mice. • Histologically treated placenta had normal cellular morphology.

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Supplementary material.

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Lalita Sharma, Geeta Shukla ⁎

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Department of Microbiology, Panjab University, Chandigarh 160014, India

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Treatment of pregnant BALB/c mice with sulphadoxine pyrimethamine or chloroquine abrogates Plasmodium berghei induced placental pathology

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Article history: Received 4 October 2012 Received in revised form 17 August 2013 Accepted 29 August 2013 Available online xxxx

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Malaria infection during pregnancy is a risk factor for foetus survival and is associated with abortion, premature delivery and low birth weight of infants in malaria endemic regions. In these regions, prophylactic measures and treatment mainly rely on chloroquine and sulphadoxine pyrimethamine, but their efficacy in reducing the placental pathology has not been studied. Therefore, the present study was designed to assess the effectiveness of chloroquine and sulphadoxine pyrimethamine treatment in reducing the placental pathology of Plasmodium berghei infected BALB/c mice. It was observed that pregnant-infected mice, treated either with chloroquine or sulphadoxine pyrimethamine had significantly lower percent parasitaemia, 100% survival and delivered normally compared with untreated pregnant-infected mice. Interestingly, antimalarial treatment significantly reduced malondialdehyde (MDA) levels, measure of lipid peroxidation and number of apoptotic cells in the placentae of pregnant-infected treated mice. Histologically also no morphological and cellular alterations were observed in the placentae of pregnant-infected treated mice. Taken together, the study shows the effectiveness of chloroquine and sulphadoxine pyrimethamine treatment, when administered in second trimester in abrogating malaria induced oxidative stress, apoptosis and histopathological alterations in the placenta, leading to normal foetal development. © 2013 Published by Elsevier Ireland Ltd.

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Keywords: Malaria Pregnancy Placenta Sulphadoxine pyrimethamine Chloroquine Malondialdehyde Superoxide dismutase Catalase

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1. Introduction

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Malaria has been recognized as one of the major public health problems, killing about 1–2 million people every year, mostly children less than five years of age and pregnant women [1,2]. In sub-Saharan Africa, malaria infection is estimated to cause 400,000 cases of severe maternal anaemia and 75,000–200,000 infant deaths annually [3–5]. Maternal malaria not only imposes an additional burden to the mother but also affects the developing foetus and is more severe in primigravidae than in multigravidae. It has also been shown that maternal malaria adversely affects the development and survival of the foetus through maternal anaemia, abortion, stillbirth and low birth weight [6–8]. The pathological alterations both in human and mouse placenta due to malaria infection results into sequestration of parasite infected RBC, recruitment of monocytes within the intervillous spaces of the placenta and syncytiotrophoblastic damage due to higher placental parasitaemia than peripheral parasitaemia [9–11]. Recently, we have reported that the altered placental pathology in Plasmodium berghei infected mice is due to enhanced oxidative stress and mitochondrial mediated pathway of apoptosis [12,13].

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⁎ Corresponding author at: Department of Microbiology, Basic Medical Sciences Building, Panjab University, Chandigarh 160014, India. Tel./fax: +91 172 2541770. E-mail address: [email protected] (G. Shukla).

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It is really a great challenge to diagnose and treat complicated malaria especially in pregnancy and deal with antimalarial associated complications like renal failure, hypoglycaemia, rapidly developing and cerebral oedema. Moreover, antimalarial treatment during pregnancy has been associated with embryotoxicity that can lead to cardiovascular malformations or skeleton abnormalities [14]. As per the recommendation of WHO, three approaches are used to reduce the burden of malaria infection during pregnancy: effective case management, insecticide treated nets (ITNs), and intermittent preventive therapy (IPT). IPT includes either two or three doses of sulphadoxine pyrimethamine (SP) that are given during the second and third trimesters, at least a month apart [15]. However, in India, chloroquine (CQ) is still the drug of choice for prevention and treatment of malaria in pregnancy, but in CQ resistant areas, a weekly regimen of CQ along with proguanil is recommended [16]. Though in India SP is in the current drug policy, SP is only used as a second line of treatment and is not readily recommended for pregnant women due to the prevalence of SP resistant strains in certain states like Assam and Orissa [17]. Although SP and CQ have been used both as prophylactic and therapeutic antimalarials for decades during pregnancy, their efficacy to reduce the placental pathology has not been studied and warrants further investigation. Therefore, the present study was designed to assess the effectiveness of sulphadoxine pyrimethamine and chloroquine treatment on the placental pathology in P. berghei infected pregnant BALB/c mice.



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2. Materials and methods

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2.1. Parasite

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P. berghei (NK 65), a lethal rodent strain was used in the study. The strain was maintained in mice by serial passage of P. berghei infected red blood cells (iRBC).

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BALB/c mice, 6–8 weeks old (20–22 g) were obtained from the central animal house of Panjab University, Chandigarh, India. These animals

Fig. 3. Efficacy of sulphadoxine pyrimethamine (SP) and chloroquine (CQ) treatment on survival rate in pregnant and non-pregnant P. berghei infected mice.

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Fig. 1. Parasitaemic levels in different groups of mice. Values are expressed as mean ± standard deviation. ≠(p b 0.05) vs. nonpregnant-infected, *(p b 0.01) vs. nonpregnantinfected, #(p b 0.01) vs. pregnant-infected.

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Female mice were housed overnight with male mice of the same strain in the ratio of 2:1 and were monitored daily both in the morning and in the evening for the presence of vaginal plug. The mice showing presence of vaginal plug were marked as first gestational day [12,13,18].

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were provided with standard pellet diet and water ad libitum. Care, use 95 and disposal of animals were done according to the guidelines of the 96 Institutional Animal Ethical Committee (44/99/CPCSEA). 97

Fig. 2. Percent reduction in parasitaemia on day 6 PI in P. berghei infected and sulphadoxine pyrimethamine (SP) or chloroquine (CQ) treated mice. Values are expressed as mean ± standard deviation.


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Table 1 Efficacy of sulphadoxine pyrimethamine and chloroquine treatment in P. berghei infected pregnant mice.

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Pregnant Pregnant-infected Pregnant-infected sulphadoxine pyrimethamine treated Pregnant-infected chloroquine treated

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The animals were divided into seven groups. Group I (nonpregnantinfected, n = 8): These animals were inoculated intraperitoneally with 1 × 106 iRBC [12,13]. Group II (nonpregnant-infected, CQ treated, n = 8): These mice were infected as in group I but were administered chloroquine (IPCA Laboratories, Mumbai, India) orally as 6.25 mg/kg body weight daily for a period of 4 days [19]. The CQ treatment was started, 2 h after malarial infection. Group III (nonpregnant-infected, SP treated, n = 8): These mice were also infected as in group I but were treated orally with single dose of sulphadoxine pyrimethamine (LUPIN Ltd. Aurangabad, India; 26 mg/kg body weight), 2 h after malarial infection [20]. Group IV (pregnant, n = 9): These pregnant animals were inoculated intraperitoneally with normal saline on GD 10 ± 2. Group V (pregnant-infected, n = 16): Pregnant mice were inoculated intraperitoneally with 1 × 106 iRBC on GD10 ± 2. Group VI (pregnant-infected,

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Percent parasitaemia in all infected groups of mice was monitored on every alternate day in Giemsa stained tail blood films by examining at least 500 cells for one month and percent survival was calculated. Mice belonging to groups IV, V and VI were sacrificed by cervical dislocation on day 6 post infection (PI) and placentae were removed for estimation of oxidants and antioxidants, DNA isolation and for ethidium bromide/acridine orange staining. For histopathological

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CQ treated, n = 16): These mice were infected as in group V but were treated orally with CQ (6.25 mg/kg body weight daily for a period of 4 days), 2 h after malarial infection [19]. Group VII (pregnant-infected, SP treated, n = 16): These mice were also infected as in group V and were treated orally with single dose of SP, 2 h after malarial infection [20].

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Fig. 4. MDA Levels (A) and catalase activity (B) in the placenta of mice belonging to various groups. Values are expressed as mean ± standard deviation. *(p b 0.05) vs. pregnant, # (p b 0.05) vs. pregnant-infected.


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studies placentae were fixed in 10% buffered formalin, processed for haematoxylin and eosin (H&E) staining and observed by light microscope.

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The amount of malondialdehyde (MDA), a measure of lipid peroxidation, was quantitated according to the method of Wills [22]. In brief, 0.5 ml of Tris–HCl buffer (0.1 M, pH 7.4) was added to 0.5 ml of tissue homogenate and incubated at 37 °C for 2 h. After incubation, 1.0 ml of 10% (w/v) chilled trichloroacetic acid was added followed by cold centrifugation at 100 × g for 10 min. To 1.0 ml of supernatant, 1.0 ml of 0.67% (w/v) thiobarbituric acid was added and kept in boiling water bath for 10 min. After cooling the tubes, 1.0 ml of distilled water was added and absorbance was measured at 532 nm. The results were expressed as nanomoles of MDA per milligram of protein, using the molar extinction coefficient of chromophore (1.56 × 105 M−1 cm−1).

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The catalase (EC 1.11.1.6) activity in post mitochondrial supernatant 155 was assayed by the method of Luck [23]. To carry out the assay 100 ml 156

2.6. Preparation of placental homogenates and post mitochondrial supernatant

Placental homogenates were prepared in phosphate buffered saline using a Potter-Elvehjem homogenizer. Placental homogenates were cold centrifuged at 8000 × g for 10 min, supernatants were labelled as post mitochondrial supernatants (PMS) and stored at −20 °C till further use. Protein concentration was measured as per Lowry et al. [21].

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Fig. 5. GSH levels (A) and SOD activity (B) in the placenta of mice belonging to various groups. Values are expressed as mean ± standard deviation.

Fig. 6. DNA fragmentation assay in the placenta of P. berghei infected mice on day 6 PI. Pregnant-infected mice showing DNA fragmentation compared to intact DNA in mice belonging to pregnant-infected SP treated, pregnant-infected CQ treated and pregnant groups.



The levels of GSH were estimated as described by Ellman [24]. Briefly, 1 ml of placental homogenate was precipitated with 1.0 ml of 4% sulphosalicyclic acid, kept at 4 °C for at least 1 h and centrifuged at 100 ×g for 15 min at 4 °C. The assay mixture contained 0.1 ml of supernatant, 0.2 ml of 0.01 M dithionitro benzoic acid (DTNB) and 2.7 ml of phosphate buffer (0.1 M, pH 8.0). Absorbance was measured at 412 nm and results were expressed as micromoles of GSH per milligram of protein.

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2.10. Assessment of super oxide dismutase (SOD) activity

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SOD (EC 1.15.1.1) activity in post mitochondrial supernatant was assayed according to the method of Kono [25]. The reaction was initiated by the addition of 0.5 ml of hydroxylamine hydrochloride to the reaction mixture containing 2.0 ml nitroblue tetrazolium (NBT) and 0.1 ml PMS. SOD activity was expressed as units of SOD per milligram of protein where one unit activity is defined as the amount of SOD required to inhibit the rate of reduction of NBT by 50%.

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Results were expressed as mean ± standard error (SE). The inter 203 group variation was assessed by two way analysis of variance 204 (ANOVA) and statistical significance was calculated at P b 0.05. 205

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Cells were isolated by teasing placentas with frosted end slides. Dispersed cell suspension was filtered using a nylon mesh and centrifuged at 100 × g for 5 min at 25 °C. RBC in the cell pellet were lysed with chilled 2% saponin and centrifuged at 100 ×g for 10 min. The sediment containing placental cells were washed thrice and finally suspended in PBS. The number of cells in the suspension was counted using a haemocytometer and isolated placental cells were observed under a fluorescence microscope after staining with ethidium bromide/acridine orange stain.

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DNA was isolated by the method described by Strauss [26] with minor modification. Briefly, 60–70 mg placental tissue was minced, suspended in 500 μl digestion buffer and kept at 50 °C overnight. An equal volume of the Tris-saturated phenol was added to the digested tissue and centrifuged at 8000 ×g for 15 min. The upper layer formed after centrifugation was subjected to phenol–chloroform–isoamylalcohol extraction procedure. Finally, DNA was precipitated with chilled ethanol, washed with 70% ethanol, dried and dissolved in Tris–EDTA buffer. Isolated DNA was electrophoresed on 1.2% agarose ethidium bromide gel and analysed by Gel Doc EZ Imager (Bio-Rad).

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of phosphate buffer (0.05 M, pH 7.2) was made and 0.16 ml of H2O2 was added. The assay mixture consisted of 3 ml of the phosphate buffer and 5 μl of PMS. Change in absorbance was read at 240 nm. The results were expressed as millimoles of H2O2 decomposed per minute per milligram of proteins using the molar extinction coefficient of the chromophore (0.0394 mM−1 cm−1).

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Fig. 7. Apoptotic cells in ethidium bromide/acridine orange (Etbr/Ar) stained placenta. Pregnant-infected mice showing higher number of apoptotic (orange nucleus) cells in the placenta compared with pregnant, pregnant-infected SP treated and pregnant-infected CQ treated groups.



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The percent parasitaemia increased gradually both in nonpregnantinfected and pregnant-infected mice and attained peak parasitaemia on day 14 PI (62%) and day 10 PI (70%) respectively, but pregnant-infected mice had significantly higher (p b 0.05) percent parasitaemia at each point of observation beginning from day 2 PI compared with nonpregnant-infected mice. Interestingly, mice belonging to nonpregnant-infected CQ treated, nonpregnant-infected SP treated, pregnant-infected CQ treated and pregnant-infected SP treated had significantly lower (p b 0.01) percent parasitaemia compared with nonpregnant-infected and pregnant-infected mice. More specifically, all the treated animals had similar pattern of parasitaemia and became parasite free by day 24 PI (Figs. 1 & 2). However, when mice were treated with a reduced dose of CQ and SP (3.5 mg/kg body weight) for a period of 9 days, the maximum parasitaemia attained by mice was only 1.2% and parasitaemia was reduced to 0% by day 10 PI, which clearly indicated the effectiveness of these antimalarial drugs (Supplementary data, Fig. S1).

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It was observed that all mice belonging to antimalarial treated groups survived compared with 100% mortality in nonpregnant-

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The catalase activity in the placenta of pregnant-infected CQ treated and pregnant-infected SP treated mice increased significantly (p b 0.05) on day 6 PI compared with pregnant-infected mice (Fig. 4B). However, levels of GSH and SOD were similar in the placentae of pregnant mice belonging to groups IV, V, VI and VII (Fig. 5A and B).

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Mice belonging to pregnant-infected CQ or SP treated groups had significantly reduced (p b 0.05) MDA levels in the placenta on day 6 PI compared with pregnant-infected mice. However, pregnantinfected mice had significantly higher (p b 0.05) MDA levels compared with pregnant mice (Fig. 4A).

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infected and pregnant-infected mice (Fig. 3). Interestingly, all pregnant-infected CQ treated and pregnant-infected SP treated mice delivered normal pups as their weights were comparable to the weights of pups delivered by pregnant mice. There was 75% mortality in pregnant-infected mice before parturition and only 25% of the pregnant-infected mice survived to deliver pups and these pups died after 2 h of delivery (Table 1).

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Fig. 8. (A) Number of apoptotic cells in the placenta of different groups of mice. Values are expressed as mean ± standard deviation. *(p b 0.05) vs. pregnant, #(p b 0.05) vs. pregnantinfected. (B) Bar diagram represents the percentage reduction in apoptotic cells in the placenta after CQ and SP treatment. Values are expressed as mean ± standard deviation.


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Histopathologically, the placenta of pregnant-infected mice showed parasitized RBC in the intervillous space of placenta and deposition of malarial pigments compared with the normal cellular morphology of placenta belonging to pregnant mice. Interestingly, SP and CQ treated pregnant mice had normal cellular placental morphology, clearly demonstrating the abrogation of placental pathology by SP or CQ treatment (Fig. 9A, B, C, D).

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Despite all the efforts made to control malaria, it is still a major public health problem in underdeveloped and developing countries especially posing a high risk to children less than five years of age and pregnant women [4]. WHO and national health agencies recommend SP and CQ as the prophylaxis during pregnancy in malaria endemic areas, to reduce maternal death and successful pregnancy outcome

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Interestingly, no DNA fragmentation was observed in the placenta of mice belonging to pregnant, pregnant-infected CQ treated and pregnantinfected SP treated groups compared with faint DNA fragments in the placenta of pregnant-infected mice (Fig. 6).

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[27,28]. Since the efficacy of SP and CQ in reducing malaria associated placental pathology with respect to oxidative stress and apoptosis has not been studied, the present study was designed. The study demonstrated that the treatment of pregnant and nonpregnant P. berghei infected mice either with SP or CQ resulted into 90% reduction in percent parasitaemia on day 6 PI and 100% survival compared with 100% mortality in pregnant-infected and nonpregnantinfected mice. The present observation is in accordance with earlier studies where 86.2% reduction in parasitaemia has been observed on day 5 PI and parasitaemia was reduced to 0% after antimalarial treatment in P. berghei infected mice [29,30]. Interestingly, all pregnant mice receiving antimalarial treatment after malarial infection delivered normally and survived till follow-up of the experiment. These observations clearly show that both SP and CQ are effective in attenuating the malarial infection which lead to normal delivery and can be used as the therapeutic agent in the late phase of pregnancy. Oxidative stress is the overabundance of reactive oxygen species (ROS) in the body which causes subsequent lipid peroxidation and has been found to be responsible for many human diseases, including malaria [31,32]. Recently, we have found that increased levels of MDA, a measure of lipid peroxidation, are responsible for the placental pathology in malarial infection [12,13]. Interestingly, in the present study we found that both SP and CQ treatments for pregnant-infected mice restored normal levels of MDA and catalase activity. However, results of catalase activity are contrary to earlier study, where a decreased activity of catalase was found in the blood of malaria patients (both male and female) treated with CQ [33]. The observed difference in catalase activity in this study may be either due to the murine model or material (placenta) used for the analysis. The antimalarial treatment further reduced the number of apoptotic cells in the placenta of infected mice as is evident by DNA ladder and ethidium bromide/acridine orange staining.

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Fig. 9. Photomicrograph of the placenta on day 6 post infection. Pregnant-infected mice showing plugging placental sinusoids with parasitized erythrocytes and malarial pigments compared with normal placental morphology in pregnant, pregnant-infected SP treated and pregnant-infected CQ treated mice (100×, H & E staining).


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The financial assistance provided by the University Grant Commission (UGC) New Delhi, India to carry out the present work is highly acknowledged.

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Appendix A. Supplementary data

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Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.parint.2013.08.016.

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Histopathologically too, the placental cell morphology was normal in pregnant treated mice. These observations clearly indicate that SP and CQ are equally effective in reducing peripheral percent parasitaemia and malaria associated placental pathology in mice particularly when used in the later phase of pregnancy. The present study clearly highlights the protective mechanism offered by SP and CQ and corroborates with earlier studies, where intermittent preventive treatment with SP greatly reduced the malarial burden in pregnant women and associated low birth weight incidence in foetuses in malaria endemic areas [34–36]. This is the first ever study providing a strong rationale about the protective efficacy of sulphadoxine pyrimethamine and chloroquine treatment in preventing the placental pathology and associated foetus loss during malaria infection due to decreased percent parasitaemia, reduced oxidative stress and apoptosis in the placenta. However, present observations need to be investigated in humans as the results may vary due to biological differences in human and murine model and presence of sulphadoxine pyrimethamine and chloroquine resistant Plasmodium falciparum strains in the community.

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