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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 8  |  Issue : 1  |  Page : 23-32

Artesunate-induced hemolysis and hypoglycemia in rats: Gender implication and role of antioxidant enzymes


1 Department of Physiology, College of Health Sciences, University of Ilorin, Ilorin, Nigeria
2 National Health Insurance Scheme, North Central A Zonal Office, Kwara State Ministry of Health Premises, Ilorin, Kwara State, Nigeria

Date of Web Publication12-Apr-2017

Correspondence Address:
Peter O Ajayi
Department of Physiology, College of Health Sciences, University of Ilorin, P.M.B. 1515, Ilorin, Kwara State
Nigeria
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/joah.joah_37_16

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  Abstract 


Introduction: The gender implication of the effects of artesunate on hematological parameters and blood glucose is not well understood. The present study investigated the effects of artesunate on some hematological parameters, blood glucose, and antioxidant enzymes in both the sexes.
Materials And Methods: A total of 35 male and 35 female Wistar rats were randomly divided into five groups (n = 7). Group one rats received distilled water; groups two and three received 2.90 mg/kg artesunate on the first day and 1.45 mg/kg artesunate from the second day until the fifth and fifteenth days, respectively; and groups four and five received 8.70 mg/kg artesunate on the first day and 4.35 mg/kg artesunate from the second day until the fifth and fifteenth days, respectively.
Results: The results of this study showed that an increase in G-6-PD activity alone prevented hemolysis in the male, but not in the female rats following a short duration of treatment. However, after a longer duration of treatment, a synergy between gucose-6-phosphate dehydrogenase (G-6-PD) and catalase prevented hemolysis in the female rats, whereas hemolysis occurred in the male rats due to the absence of an increased G-6-PD activity. Probably due to hormonal differences, artesunate reduced fasting blood glucose in the females, but it was either hypoglycemic (at low dose) or hyperglycemic (at high dose) in the male rats.
Discussion: In conclusion, the present study suggests that a duration-dependent difference exists in the onset of artesunate-induced hemolysis and the response of antioxidant enzymes in the male and female rats and that following longer duration of treatment, hypoglycemia could be sustained in the female rats while counterregulatory mechanism(s) could be triggered in the male rats.

Keywords: Antioxidants, artesunate, free radicals, hemolysis, hypoglycemia


How to cite this article:
Salman MT, Ajayi PO, Alagbonsi AI, Olatunji LA. Artesunate-induced hemolysis and hypoglycemia in rats: Gender implication and role of antioxidant enzymes. J Appl Hematol 2017;8:23-32

How to cite this URL:
Salman MT, Ajayi PO, Alagbonsi AI, Olatunji LA. Artesunate-induced hemolysis and hypoglycemia in rats: Gender implication and role of antioxidant enzymes. J Appl Hematol [serial online] 2017 [cited 2017 May 25];8:23-32. Available from: http://www.jahjournal.org/text.asp?2017/8/1/23/204423




  Introduction Top


Matured red blood cells (RBCs) contain large amounts of iron, lack nucleus and mitochondria, lack ability to synthesize new proteins, have high rates of degradation of detoxifying enzymes, and operate in highly oxygenated tissues, making them uniquely and continuously vulnerable to oxidative stress.[1],[2] The oxidative stress and consequent hemolysis of RBCs are prevented via Nicotinamide adenine dinucleotide phosphate-catalase-glutathione peroxidase pathway by gucose-6-phosphate dehydrogenase (G-6-PD), a rate-limiting enzyme in the pentose phosphate pathway.[1],[3] G-6-PD deficiency leads to drug-dependent (e.g., antimalarial drugs) and −independent hemolytic anemia.[3] In addition to G-6-PD, the body has other protective mechanisms against oxidation, and these include prevention of formation of reactive oxygen species (ROS), scavenging of various forms of ROS, and repair of oxidized cellular contents.[1] In general, a partial defect in any of these antioxidant defense systems can harm the RBCs and promote senescence but is without chronic hemolytic complaints.

Although, several drugs are available for the treatment of malaria, their efficacy has been limited. Treatment failures have been linked majorly to the development of resistance to the existing antimalarial agents by the malaria parasites,[4],[5],[6],[7] and this has created a need for new drugs.[8] Discovery of artemisinin (first line of treatment for severe malaria) and its derivatives has given a renewed hope for combating against resistant malaria. It generates free radicals, thus, alkylating the parasite’s membrane.[9] On the basis of the clinical trials, artesunate produces fewer life-threatening side effects than quinine, with fewer recorded incidences of low blood sugar (hypoglycemia) and anemia.[10] The World Health Organization guidelines recommended intravenous artesunate as the first-line therapy for severe falciparum malaria, yet many patients, especially from low economic status, can only afford the less expensive but effective oral artesunate. The mechanism of artesunate action on the erythrocytes remains poorly known, although Salman[11] implicated G-6-PD, which was reportedly increased in the nonmalaria-infected and artesunate-treated erythrocytes in the male rats. Another researcher also observed an increase in G-6-PD in the malarial-infected erythrocytes of the patients treated with artesunate.[12] In their work on the male rats administered with artesunate, Shittu et al.[13] reported a significant increase in malondialdehyde (MDA) but with a simultaneous, significant decrease in antioxidant activities, especially of superoxide dismutase (SOD). In conformity, another study reported that G-6-PD overexpression decreased endothelial cell oxidant stress.[14]

Serious concern has been raised about uncontrolled use of artemisinin derivatives in malaria endemic areas such as Nigeria. Moreover, self-medication and purchase of antimalarial drugs in the open market are rampant.[15] The possibility of overdose administration and misappropriation in the usage of antimalarial agents are very common, all of which could lead to toxic effects of the drugs.[16],[17],[18] While some researchers showed that artesunate (via oxidative actions) will have the possibility of causing spermatotoxicity, neurotoxicity, hepatotoxicity, and hemotoxicity, others showed that artesunate is cardioprotective and erythroprotective and decreases endothelial cell oxidant stress.[6],[11],[14],[18],[19]

The response of antioxidant enzymes to the effects of artesunate on the erythrocytes remains poorly known, although an increased G-6-PD activity was reported in nonmalarial and malaria-infected RBCs of the male rats.[14],[20] Although the contribution of other antioxidant enzymes to the protection against artesunate-associated hemolysis is not fully understood, there is no information on the effect of artesunate on the hematological parameters in the female rats, which this study sought to provide.


  Materials and Methods Top


Animals

Male and female albino rats (140–180 g) were obtained from a reputable breeder in Ilorin and acclimatized in the Central Animal House, College of Health Sciences, University of Ilorin, Ilorin for three weeks. They were provided with standard diet and water ad libitum and maintained at room temperature in a day–night cycle. The “Principles of Laboratory Animal Care” (NIH Publication No. 85-23, revised 1985) were followed. All the experiments had been examined and approved by our Institutional Ethics Committee.

Experimental design

A total of 35 male and 35 female rats were separately divided in a blinded fashion into five groups (n = 7 rats each) and treated as follows:



The artesunate was freshly prepared on a daily basis to avoid contamination and was administered by oral gavage with the use of a cannula. The doses of treatment were chosen to mimic those in humans (groups two and three) and also tripled (groups four and five). The durations for treatment were also chosen to mimic those in humans (groups two and four) but also tripled (groups three and five). The animals were fasted for about 18 h at the end of the administration for the determination of fasting blood sugar just before they were sacrificed by decapitation under pentobarbitone anesthesia, 37 mg/kg i.p.[21] Blood samples were then collected for analysis.

Hematological analysis

The whole blood was collected from the animals into EDTA bottle and assayed for the RBCs count, packed cell volume (PCV), and hemoglobin (Hb), using standard laboratory techniques.

Determination of malondialdehyde concentration

The assay method of Hunter et al.[22] modified by Gutteridge and Wilkins[23] was utilized.

Determination of superoxide dismutase activity

The method described by Misra and Fridovich[24] was used for this analysis.

Determination of catalase activity

The catalase (CAT) activity was determined spectrophotometrically according to the standard protocol, as described by Clariborne method.[25]

Glucose-6-phosphate dehydrogenase activity

G-6-PD activity was estimated as previously described.[26]

Blood glucose estimation

Blood glucose was determined by the glucose oxidase method using a glucometer.[27]

Statistical analysis

Data were expressed as means ± standard error of mean (SEM) and analyzed with one-way analysis of variance, followed by a post hoc least significant difference for multiple comparison using Graphpad Prism 5 software (San Diego, California, USA). P-values less than or equal to 0.05 were considered statistically significant.


  Results Top


Effects of artesunate on hematological parameters in the male and female rats

There was no significant change in the RBCs following a short-period (5 days) administration of LDA (5.32 ± 0.14 millions/mm3; P > 0.05) or HDA (5.74 ± 0.33 millions/mm3; P > 0.05) to the male rats, but was significantly reduced by a long-period (14 days) administration of either LDA (4.43 ± 0.30 millions/mm3; P < 0.001) or HDA (5.10 ± 0.10; P < 0.05) when compared to the control (5.77 ± 0.22 millions/mm3). However, the RBC in the female rats was significantly reduced by a short-period administration of LDA (4.74 ± 0.11 millions/mm3; P < 0.05) or HDA (4.50 ± 0.04 millions/mm3; P < 0.001), whereas there was a slight increase in the RBC count after the long-period administration of either LDA (5.09 ± 0.13 millions/mm3; P > 0.05) or HDA (5.41 ± 0.08 millions/mm3; P < 0.01) when compared to the control (5.00 ± 0.60 millions/mm3). In addition, the RBC counts in the female rats that received placebo or either dose of artesunate for a short duration was lower when compared with their male counterpart. However, after a long duration of treatment with HDA, the RBC count was higher in the female rats when compared to their counterpart male rats that received similar treatments [Figure 1].
Figure 1: Effects of artesunate on the red blood cell (RBC) count in the male and female rats. Values are expressed as mean ± SEM (n = 7). *P < 0.05, **P < 0.01, ***P < 0.001 versus control within each gender; #P < 0.05, ##P < 0.01 versus similar treatment in male

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PCV in the male rats was not affected by a short-period administration of LDA (41.1 ± 0.38; P > 0.05) or HDA (41.2 ± 0.32%; P > 0.05), but was reduced by a long-period administration of either LDA (27.8 ± 0.51%; P < 0.001) or HDA (40.1 ± 0.23%; P > 0.05) when compared to the control (41.0 ± 0.56%). However, PCV in the female rats was significantly reduced by a short-period administration of LDA (34.6 ± 1.60%; P < 0.001) or HDA (32.3 ± 0.58%; P < 0.001) and a long-period administration of HDA (37.6 ± 0.87%; P < 0.01), while there was no significant change after a long-period administration of LDA (43.00 ± 0.80%; P > 0.05) when compared to the control (41.1 ± 0.50%). Though the control male and female rats showed no difference in PCV, all other treatments but LDA given for a long-period significantly reduced PCV in the female rats when compared to the male rats that received similar treatments [Figure 2].
Figure 2: Effects of artesunate on packed cell volume (PCV) in the male and female rats. Values are expressed as mean ± SEM (n = 7). **P < 0.01, ***P < 0.001 versus control within each gender; #P < 0.05, ###P < 0.001 versus similar treatment in male

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Similarly, Hb concentration in the male rats was not affected by a short-period administration of LDA (12.88 ± 0.25 g/dl; P > 0.05) or HDA (12.86 ± 0.37 g/dl; P > 0.05), but was reduced by a long-period administration of either LDA (9.04 ± 0.21 g/dl; P < 0.001) or HDA (13.03 ± 0.37 g/dl; P > 0.05) when compared to the control (13.26 ± 0.26 g/dl). The reductions in the Hb concentration in the female rats treated for a short-period was only significant with LDA (11.54 ± 0.49 g/dl; P < 0.05) but not with HDA (12.02 ± 0.35 g/dl; P > 0.05), while a long-period treatment with either LDA (12.83 ± 0.33 g/dl; P > 0.05) or HDA (11.87 ± 0.29 g/dl; P > 0.05) had no significant effect on Hb concentration when compared to the control (12.74 ± 0.30 g/dl). All other treatments, but LDA for long duration and HDA for a short period, reduced Hb concentration in the female rats when compared to the male rats that received similar treatments [Figure 3].
Figure 3: Effects of artesunate on hemoglobin concentration in the male and female rats. Values are expressed as mean ± SEM (n = 7). *P < 0.5, ***P < 0.001 versus control within each gender; #P < 0.05, ###P < 0.001 versus similar treatment in male

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Effects of artesunate on lipid peroxidation and antioxidant enzymes in the male and female rats

Except in those that received LDA for a long-period (1.58 ± 0.02 nM/ml; P > 0.05), MDA concentration was significantly increased in the male rats that received LDA for a short-period (1.68 ± 0.04 nM/ml; P < 0.01) and in those that received HDA for a short-period (1.69 ± 0.05 nM/ml; P < 0.01) and a long-period (1.81 ± 0.08 nM/ml; P < 0.001) when compared to the control (1.46 ± 0.02 nM/ml). Similarly, MDA concentration was significantly increased in the female rats treated with LDA (4.93 ± 0.18 nM/ml; P < 0.001) and HDA (2.24 ± 0.02 nM/ml; P < 0.001) for a short duration, while a long-period administration of either LDA (1.63 ± 0.06 nM/ml; P > 0.05) or HDA (1.55 ± 0.01 nM/ml; P > 0.01) did not have any significant effect on MDA concentration when compared to the control (1.69 ± 0.06 nM/ml). Treatment of the female rats with HDA and LDA for a short-period significantly (P < 0.001) increased MDA concentration when compared with the male rats that received similar treatments [Figure 4].
Figure 4: Effects of artesunate on malondialdehyde level (MDA) in the male and female rats. Values are expressed as mean ± SEM (n = 7). **P < 0.01, ***P < 0.001 versus control within each gender; ##P < 0.01, ###P < 0.001 versus similar treatment in male

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SOD in the male rats was significantly reduced by a short-period administration of LDA (17.35 ± 0.14 U/ml; P < 0.05) and HDA (17.06 ± 0.30 U/ml; P < 0.01), but was significantly increased by a long-period administration of LDA (19.26 ± 0.12 U/ml; P < 0.001) and HDA (18.55 ± 0.18 U/ml; P < 0.05) when compared to the control (17.94 ± 0.22 U/ml). Similarly, in the female rats, SOD activity was significantly reduced by a short-period administration of LDA (14.27 ± 0.43 U/ml, P < 0.001) and HDA (14.46 ± 0.72 U/ml; P < 0.001), but the slight increase caused by a long-period administration of LDA (17.75 ± 0.17 U/ml; P > 0.05) and HDA (18.64 ± 0.51 U/ml; P > 0.05) was not significant when compared to the control (17.68 ± 0.35 U/ml). Except in the rats that received HDA for a long period, the SOD in other female rats are lower than in the male rats that received similar treatments [Figure 5].
Figure 5: Effects of artesunate on superoxide dismutase activity (SOD) in the male and female rats. Values are expressed as mean ± SEM (n = 7). *P < 0.05, **P < 0.01, ***P < 0.001 versus control within each gender; ##P < 0.05, ###P < 0.01 versus similar treatment in male

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There was no significant change (P > 0.05) in the CAT activity in all treatment groups of the male rats (162.23 ± 0.27, 162.03 ± 0.29, and 162.78 ± 0.00 U/ml), except in those that received HDA for a long duration (164.00 ± 0.99 U/ml; P < 0.05) when compared to the control (162.50 ± 0.01 U/ml). However, CAT activity in the female rats was reduced after a short-period treatment with LDA (163.00 ± 0.79 U/ml; P < 0.001) and HDA (163.73 ± 0.78 U/ml; P < 0.001) but significantly increased after a long-period administration of LDA (183.96 ± 0.30 U/ml; P < 0.001) and HDA ((80.65 ± 0.48 U/ml; P < 0.001) when compared to the control (173.52 ± 1.08 U/ml). The CAT activity in the female rats that served as the control and those that received LDA and HDA for a long duration was higher than those observed in the male rats that received similar treatments [Figure 6].
Figure 6: Effects of artesunate on catalase activity in the male and female rats. Values are expressed as mean ± SEM (n = 7). *P < 0.05, ***P < 0.001 versus control within each gender; ###P < 0.001 versus similar treatment in male

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The glucose-6-phosphate dehydrogenase (G-6-PD) activity was significantly higher in the male rats that received a short-period treatment with LDA (482.66 ± 8.39 mU/109 erythrocytes; P < 0.001) and HDA (486.46 ± 18.53 mU/109 erythrocytes; P < 0.001) while a long-period treatment with LDA (344.25 ± 8.73 mU/109 erythrocytes) and HDA (342.13 ± 4.86 mU/109 erythrocytes) also showed a slight but insignificant (P > 0.05) increase when compared with the control (326.24 ± 11.37 mU/109 erythrocytes). Similarly, in the female rats, a short-period treatment with LDA (276.33 ± 2.01 mU/109 erythrocytes; P < 0.001) and HDA (183.32 ± 0.73 mU/109 erythrocytes; P > 0.05) and a long-period treatment with LDA (277.12 ± 4.91; P < 0.001) increased the G-6-PD, while a long-period treatment with HDA [155.59 ± 0.53 mU/109 erythrocytes; P < 0.001) reduced the G-6-PD when compared to the control (177.54 ± 1.49 mU/109 erythrocytes). However, the G-6-PD in the female rats was significantly lower than that in the male rats that received similar treatments [Figure 7].
Figure 7: Effects of artesunate on glucose-6-phosphate dehydrogenase (G-6-PD) in the male and female rats. Values are expressed as mean ± SEM (n = 7). ***P < 0.001 versus control within each gender; ###P < 0.001 versus similar treatment in male

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Effects of artesunate on the blood glucose levels in the male and female rats

Blood glucose level in the male rats was reduced by the LDA (61.7 ± 1.46 mg/dl; P < 0.001) but increased by the HDA (78.8 ± 0.85 mg/dl; P < 0.001) treatments for a short duration, while the increase caused by a long-duration administration was only significant with the HDA (81.5 ± 0.64 mg/dl; P < 0.001) but not with the LDA (74.1 ± 1.22 mg/dl; P > 0.05) when compared to the control (71.9 ± 0.48 mg/dl). However, both the LDA (73.6 ± 0.91 mg/dl, P < 0.001; 77.5 ± 0.40 mg/dl, P < 0.001) and the HDA (77.9 ± 0.98 mg/dl, P < 0.001; 76.9 ± 1.05 mg/dl, P < 0.001) administered for short duration and long duration respectively caused significant reductions in blood glucose level in the female rats when compared to the control (82.3 ± 0.47 mg/dl). Blood glucose levels in the LDA treatment for both durations were significantly higher in the female than that in the male rats, while HDA administered for long duration was significantly lower in the female than that in the male rats [Table 1] and [Figure 8].
Table 1: Effects of artesunate on blood glucose level in the male and female rats

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Figure 8: Effects of artesunate on blood glucose level in male and female rats. Values are expressed as mean ± SEM (n = 7). ***P < 0.001 versus control within each gender; #P < 0.05, ##P < 0.01, ###P < 0.01 versus similar treatment in male

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  Discussion Top


Artesunate has been shown to accumulate in three major tissues namely: the erythrocytes, the liver, and to a lesser extent, the kidney.[28] Part of the mechanisms of artesunate’s action after its accumulation in the erythrocytes is the splitting of its endoperoxide bridge by iron heme species present in the red cells and consequent release of ROS.[29] Although moderate level of ROS has beneficial effects, its excessive generation can be deleterious to the body cells, especially the erythrocytes that host Plasmodium falciparum.[3] The artesunate-induced lipid peroxidation observed with LDA treatment for a short duration in both the genders is similar to previous studies and indicates that artesunate-related hemolysis is mediated by free radical generation.[13],[19]

SOD is known as an antioxidant enzyme that scavenges superoxide anion radical into hydrogen peroxide, which in turn, is transformed to water and oxygen by yet other antioxidant enzymes such as CAT and glutathione peroxidase and are together considered as the first line of antioxidant defense enzymes against free radical-induced toxicity.[3],[30] A previous study has also reported a reduction in SOD activity in the artesunate-treated male rats similar to the observation in this study.[13] In the present study, the reduction in SOD activity in both the genders (and CAT in females), but increase in G-6-PD activity when lipid peroxidation was enhanced, is an indication of the fact that the antioxidant system switched to the second line of defense (G-6-PD), when the first line antioxidant defense enzymes were exhausted. Similar increase in G-6-PD activity had been reported by Salman[11] in artesunate-treated male rats with similar dosage, and an insignificant increase was also reported by other researchers, in malarial-infected human red cells, in which artesunate was used in a combination therapy.[20]

The RBCs are uniquely vulnerable to oxidative stress due to their continuous exposure to high concentrations of oxygen radicals, lack of nucleus and mitochondria, inability to synthesize new proteins, and degradation of detoxifying enzymes.[2] Overproduction of free radicals beyond a threshold for proper antioxidant neutralization causes redox imbalance and oxidative stress.[31] It is noteworthy that the defensive role played by G-6-PD prevented hemolysis in the male rats but could not prevent it in the female rats. This is possibly due to the fact that the extent to which artesunate induced lipid peroxidation was not as much in the male rats as in the female rats, which made free radical to be adequately scavenged by G-6-PD, hence preventing hemolysis in the male but not in the female rats.

The G-6-PD is an important house-keeping enzyme that catalyzes the first step of the pentose phosphate pathway, which is the sole source for the production of reducing capacity in the form of NADPH in the erythrocytes as erythrocytes lack the nucleus, the ribosomes, and the mitochondria (at maturity) that generate NADPH as in other cells.[32],[33] The NADPH is needed to activate glutathione reductase and CAT, which are also antioxidant enzymes and are abundant in RBCs. Acute anemia induced by antimalarial drug is an example of adverse drug reaction that has been recognized for a long time in G-6-PD-deficient people.[3] Deficiency of G-6-PD, an X-linked genetic disorder affecting more number of male (3% of neonates) than female (0.9% of neonates) has been found in the human gene containing 13 exons and mapped to Xq28 region.[34]

It is noteworthy that artesunate caused hemolysis in the female but not in the male rats even though there was an increase in G-6-PD activity in both the male and the female rats after short duration of treatment. The hemolysis in the female rats despite an increase in G-6-PD activity was probably due to the reduction in the activities of CAT and SOD. This suggests that an increase in G-6-PD activity alone is capable of preventing hemolysis in the male but not female rats when LDA was administered for a short duration. The absence of hemolysis in the female rats after a long duration of treatment, when there was an increase in both G-6-PD and CAT activities also suggests that a synergy between two or more antioxidant enzymes might be required to prevent hemolysis in the female rats treated with artesunate. Thus, the absence of this synergy between G-6-PD and CAT was probably responsible for the hemolysis in the female rats after a short duration of treatment in the female rats. The present study, therefore, suggests that an increase in G-6-PD activity alone could prevent hemolysis in the male but not female rats following a short duration of treatment with artesunate and that a synergy between G-6-PD and CAT is required to prevent hemolysis in the female rats following a long-term treatment with artesunate.

The absence of an increase in G-6-PD activity observed in the male rats with prolonged treatment as opposed to its significant increase after short-term treatment in the male rats might be linked to consequential red cells hemolyses. Although the study was not extended longer than 24-h for post-treatment/delayed hemolyses (often evident about a month after IV-artesunate treatment in human), there was significant reduction (P < 0.05) of G-6-PD at the end of the longer durations of both doses, compared to their shorter durations. Drug-induced G-6-PD deficiency and its hemizygosity can be investigated only if longer period is allowed for estimation of delayed hemolysis. Paradoxically, while artesunate-induced increase in G-6-PD activity alone prevented hemolysis at short duration, increased SOD activity alone could not prevent the hemolysis induced by a long duration of treatment with both doses. This finding suggests that G-6-PD is more crucial than SOD and CAT for the protection of the RBC in the male rats. It also suggests that an increase in the activity of G-6-PD alone can prevent hemolysis in the male rats during the short duration of treatment in addition to generating NADPH to activate the chain-braking antioxidants like SOD to minimize the damaging effects of ROS during a long duration of treatment when G-6-PD is apparently exhausted.

The artesunate-induced hemolysis observed in this study due to the absence of an increase in G-6-PD activity after long duration of treatment in the male rats is consistent with previous reports that G-6-PD deficiency caused hemolysis after administration of antimalarial drugs such as chloroquine and primaquine.[35] Many drugs, as well as fava [favism], can cause G-6-PD deficiency-related hemolysis in a reaction with oxyhemoglobin, which forms superoxide that decomposes to hydrogen peroxide.[3] Accumulation of peroxides following reduced G-6-PD results in Hb denaturation and binding to the cell membrane, seen morphologically as Heinz bodies.[3] Hemolysis occurs, when the erythrocytes pass through the spleen and the Heinz bodies are removed along with a portion of the cell membrane. After several passes through the spleen, the cell membrane loses competency and the erythrocyte is destroyed.[3]

Indeed, it is very clear from this study that the response of G-6-PD to artesunate-induced hemolysis occurred at different times in the male and the female rats. For instance, the male rats responded by increasing G-6-PD activity after a short duration of treatment with artesunate, whereas there was no such response in the female rats. Contrarily, the female rats also responded by increasing G-6-PD activity after a long duration of treatment while G-6-PD activity returned to normal in the male rats at that point in time. This probably suggests that a difference exists in the onset of the response of G-6-PD to ROS generated by artesunate and consequently a difference in the onset of artesunate-induced hemolysis in the male and the female rats. Thus, while the response of G-6-PD appears to be rapid in the male rats, the onset of hemolysis was delayed and vice versa in the female rats. The reason for this gender variation in the response of G-6-PD is not yet clear and may require further investigation. It is important to suggest the likelihood of G-6-PD deficiency as being linked to the observed hemolyses, in the female rats, especially at longer duration of HDA. Females have paired X-chromosomes, which can either have one abnormal (heterozygous) or two abnormal (homozygous) genes for G-6-PD deficiency.[36] Whether or not artesunate is capable of inducing homozygous/heterozygous G-6-PD deficiency will only become evident if further studies aiming at measuring G-6-PD deficiency alongside the markers of hemolysis such as RBC count, Hb, resticulocyte count, lactate dehydrogenase, direct bilirubin, and indirect bilirubin are conducted.

The slight but a significant increase in the RBC count in the female rats following a long duration of treatment with HDA could probably be due to stimulation of erythropoiesis in response to artesunate-induced hemolysis, which could have occurred after 5 days of treatment as evidenced by the reduction in the RBC count in the female rats treated with the same dose for 5 days.

Of great interest is the reduction in blood glucose observed following treatment with artesunate in this study. The effects of many antimalarial drugs on blood glucose as well as their mechanism of action have been well documented. For instance, the cinchona alkaloids (quinine and quinidine) are known to increase insulin secretion by blocking ATP-sensitive potassium (K+ATP) channels in pancreatic beta cells, a property shared with the sulphonylurea drugs used in the treatment of non-insulin-dependent diabetes mellitus.[37],[38] Similarly, halofantrine and chloroquine have been shown to induce an increase in plasma insulin and glucose uptake and, consequently, hypoglycemia.[39],[40] Thus, hypoglycemic effect mediated by an increase in insulin secretion due to the blocking of ATP-sensitive potassium (K+ATP) channels in pancreatic beta cells appears to be a common property shared by these antimalarial agents.

However, the artemisinin derivatives have been reported not to be associated with hypoglycemia and the proportions of the patients with hypoglycemia at study entry (8%) and during artemether treatment (11%) in adult patients with severe malaria were similar.[41] Contrarily, it was also reported that if these compounds were given to animals in high doses, it was again possible that they also block pancreatic-β cell K+ATP channels and increase insulin secretion.[42]

In this study, however, while artesunate (another artemisinin derivative) produced a blood glucose-lowering effect in all female rats at both low and high doses, it had differential effects in the male rats; reduction in blood glucose with LDA treatment for a short duration but increase in blood glucose at both durations of HDA. While the decrease in blood glucose in this study is consistent with previous studies,[37],[38],[39],[40] the observed increase in blood glucose in the male rats treated with HDA is quite contrary to the earlier report that the artemisinin derivatives such as artemether may be associated with hypoglycemia at high doses.[41],[42] Although the mechanism involved in artesunate-induced reduction/increase in blood glucose has not been investigated in this study, it is not unreasonable to suggest that artesunate might also be acting through the mechanism common to the above-mentioned antimalarial drugs to cause a reduction in blood glucose. That is, by an increase in insulin secretion due to the blocking of K+ATP channels in pancreatic-β cells.

The mechanism involved in artesunate-induced increase in blood glucose following treatment with HDA in the male but not female rats is not yet clear. However, it could be due to the release of cortisol and adrenaline, which are considered as typical stress hormones that are released via activation of the hypothalamic–pituitary–adrenal (HPA) axis under stress.[43] A previous study also observed a correlation between the plasma levels of stress hormones (cortisol and adrenaline) and oxidative stress biomarkers such as ROS and MDA.[44] Additionally, activation of the HPA axis is associated with acceleration in oxidative stress via unbalanced redox, including excessive production of mitochondrial ROS.[45],[46] A critical look at the data from this study shows that MDA concentration, which was very high when LDA was given to female rats for a short duration, became low when HDA was given. Contrarily, the MDA concentration remained significantly high in the male rats given a HDA. Thus, the sustained increase in MDA concentration in the male rats given HDA could have stimulated the release of cortisol and adrenaline leading to an increase in blood glucose.

The apparent but insignificant increase in blood glucose in the male rats treated with LDA for long duration could be due to an ongoing counter-regulatory response to the reduction in blood glucose that could have occurred earlier during the treatment as evidenced by the reduction in blood glucose in the male rats treated with the same dose for a short duration. A similar observation of a reduction and an increase in blood glucose after one week and two weeks of treatment, respectively, with aqueous extract of Telfairia occidentalis had been reported in the male rats.[47]

The question that readily comes to mind is, what other factor(s) could be responsible for the sustained reduction in blood glucose in the female rats given a HDA as opposed to the increase observed in the male rats? Could it be due to the influence of the female reproductive hormone(s) such as estrogen? A previous study has reported a decrease in fasting levels of glucose upon the administration of oral estrogen replacement in postmenopausal women.[48] In animal studies, the main steroids of the ovary, the estrogens and the progestins, have been shown to provide a protective influence to the susceptibility to experimental diabetes.[49],[50]

Furthermore, an increase in basal glycemia and an impaired glucose tolerance have been observed in ovariectomized mice as well as rats; steroid replacement experiments indicated that a deficiency of estrogens is mainly responsible for the deterioration of glucose tolerance.[51],[52] Transdermal estradiol replacement therapy in estrogen-deficient postmenopausal women was shown to improve beta-cell function in vivo and to augment insulin secretion in response to an acute glucose challenge.[53],[54] This effect was proposed to involve a tropic action of estradiol on pancreatic islets in combination with an increase in glucose transport in the muscle and an inhibition of gluconeogenesis.[54] Indeed, the islets of Langerhans have been demonstrated to express estrogen receptors[52] and to show a tropic response to estradiol treatment in vivo.[59] Therefore, if both artesunate and estrogen can stimulate insulin secretion, the synergestic effects of both can cause a very significant increase in plasma insulin leading to sustained reduction in blood glucose. Further studies on the influence of artesunate on hormones (reproductive and metabolic) and enzymes of glucose metabolism may, therefore, provide an insight into the mechanism of artesunate-induced reduction/increase in blood glucose observed in this study.

It is also noteworthy that the significant increase in blood glucose in the male rats treated with HDA could also have contributed to the inactivation of CAT, increase in MDA concentration and consequently hemolysis seen in these rats. This is consistent with an earlier report that hyperglycemia of diabetes reduced CAT activity,[55] and the assumption that hyperglycemia may be involved in the enhancement of plasma-free radical production.[56] Glucose autoxidation represents a pathway by which glucose itself generates free oxygen radicals. The enediol form of glucose may be autoxidized to an enediol radical anion. The reduced oxygen products are superoxide radical anions (O2), the hydroxyl radical (OH), and hydrogen peroxide (H2O2). The hydroxyl radicals produced by glucose autoxidation were shown to damage proteins. Moreover, an elevated glucose level was found to activate lipoxygenase enzymes[57] and a decrease in the activity of nitric oxide synthase and glutathione reductase, resulting in an increased susceptibility of the endothelial cells to damage.[48]

The present study, therefore, suggests that the use of artesunate by the patients (especially females) with symptoms of anemia may need to be handled with caution and that doses/durations should be relatively moderate as possibility of hemolysis can become evident at longer duration of oral artesunate use, as observed with this study. Co-administration of artesunate with exogenous antioxidants such as vitamins C and E is also strongly recommended. Artesunate-induced hemolysis/anemia and hypoglycemia should also be emphasized to potential drug users, abusers, and addicts, as possible side effects.

In conclusion, the present study suggests that artesunate could cause duration-dependent hemolysis and hypoglycemia probably via free radical generation, inactivation of antioxidant enzymes (CAT and SOD), and stimulation of insulin secretion. It also suggests that G-6-PD plays a very crucial role in the prevention of artesunate-induced hemolysis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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