Chronic Exposure to Arsenic in Drinking Water Can Lead to Resistance to Antimonial Drugs in a Mouse Model of Visceral Leishmaniasis

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Chronic exposure to arsenic in drinking water can lead to resistance to antimonial drugs in a mouse model of visceral leishmaniasis

Meghan R. Perrya, Susan Wylliea, Andrea Raabb, Joerg Feldmannb, and Alan H. Fairlamba,1

aDivision of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom; and bCollege of Physical Sciences–Chemistry, Trace Element Speciation Laboratory, University of Aberdeen, Aberdeen AB24 3UE, Scotland, United Kingdom

Edited by Thomas E. Wellems, National Institutes of Health, Bethesda, MD, and approved October 1, 2013 (received for review June 18, 2013) The Indian subcontinent is the only region where arsenic contam- groundwater accessed by these wells (8). The elements antimony ination of drinking water coexists with widespread resistance to and arsenic have a long therapeutic history, are closely related in antimonial drugs that are used to treat the parasitic disease the periodic table, and share many similar chemical properties visceral leishmaniasis. We have previously proposed that selection (9). In the 1980s, when Leishmania antimonial resistance was for parasite resistance within visceral leishmaniasis patients who first suspected, parasitologists used stepwise exposure to trivalent have been exposed to trivalent arsenic results in cross-resistance arsenic in the laboratory to induce antimonial cross-resistance and to the related metalloid antimony, present in the pentavalent state study mechanisms of resistance (10). Our hypothesis is that a as a complex in drugs such as sodium stibogluconate (Pentostam) similar selection process could occur in VL patients who have and meglumine antimonate (Glucantime). To test this hypothesis, been chronically exposed to environmental arsenic, such that Leishmania donovani wasseriallypassagedinmiceexposedtoar- selection for parasite resistance to arsenic would result in cross- senic in drinking water at environmentally relevant levels (10 or 100 resistance to antimonial drugs. ppm). Arsenic accumulation in organs and other tissues was pro- The incidence of arsenic contamination, VL, and antimonial portional to the level of exposure and similar to that previously resistance from all of the available data from surveys, journals, reported in human liver biopsies. After five monthly passages in mice and Web sites was previously collated by our group (7). These exposedtoarsenic,isolatedparasites were found to be completely data showed that in 10 out of 38 districts in Bihar, arsenic ex- MICROBIOLOGY − refractory to 500 μg·mL 1 Pentostam compared with the control pas- posure, endemic VL, and reported antimonial resistance coexist. − sage group (38.5 μg·mL 1) cultured in vitro in mouse peritoneal mac- Thus, in these areas, arsenic contamination of the groundwater rophages. Reassessment of resistant parasites following further has the potential to contribute to the development of Leishmania passage for 4 mo in mice without arsenic exposure showed that parasite antimonial resistance, giving epidemiological plausibility resistance was stable. Treatment of infected mice with Pentostam to the hypothesis. confirmed that resistance observed in vitro also occurred in vivo. In this study we test our hypothesis using a mouse model of We conclude that arsenic contamination may have played a signifi- arsenic exposure and chronic VL. Our findings demonstrate that cant role in the development of Leishmania antimonial resistance Leishmania parasites resistant to pentavalent antimonials can be in Bihar because inadequate treatment with antimonial drugs is not created through oral exposure to arsenic in drinking water of exclusive to India, whereas widespread antimonial resistance is. mice infected with L. donovani.

drug resistance | sodium antimony gluconate | treatment failure | Results environmental pollution Quantitation of Arsenic Exposure in BALB/c Mice by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). The hypothesis that Leishmania isceral leishmaniasis (VL) is a systemic illness caused by the parasites could become resistant to antimonial preparations Vobligate intracellular protozoan parasites Leishmania don- through exposure to arsenic within their host relies on the presence ovani and Leishmania infantum. The parasite, in amastigote form, multiplies within macrophages of the spleen, liver, and bone mar- Significance row causing fever, anorexia, weight loss, and hepatosplenomegaly (1). Effective chemotherapy for this condition is essential because Bihar, India, is the only region where arsenic contamination of untreated VL is fatal and accounts for 41,000 deaths per year (2). drinking water coexists alongside endemic visceral leishmani- Currently, there are four main antileishmanial drugs available: asis and where widespread resistance to pentavalent antimo- antimonial preparations, amphotericin B, miltefosine, and paro- nial drugs (e.g., Pentostam) occurs. We have proposed that momycin (3). Antimonial preparations have been used for almost selection for parasite resistance in infected individuals exposed a century and remain an essential part of the treatment of VL in to environmental arsenic results in cross-resistance to the related South America and sub-Saharan Africa (2). However, by the end metalloid antimony. To test this hypothesis, we have serially of the twentieth century, their efficacy in Bihar, which houses 90% passaged Leishmania donovani in mice receiving arsenic in of India’s large VL burden (4), had decreased to cure rates of less drinking water at environmentally relevant levels. In support of than 50% (5). Consequently, use of antimonial drugs is no longer this hypothesis, we show that after five monthly passages, recommended in the Indian subcontinent (6). Leishmania parasites become stably resistant to Pentostam in The underlying reasons for this epidemic of resistance in Bihar vitro and in vivo. are not fully understood. In our previous publication we proposed that the presence of arsenic in drinking water in Bihar has Author contributions: M.R.P., S.W., J.F., and A.H.F. designed research; M.R.P., S.W., and A.R. contributed to the gradual decline in efficacy of antimonial performed research; M.R.P., S.W., A.R., J.F., and A.H.F. analyzed data; and M.R.P., S.W., A.R., preparations for VL in this region (7). Subsequent to the 1970s, J.F., and A.H.F. wrote the paper. when there was a large-scale insertion of shallow tube wells The authors declare no conflict of interest. to provide clean drinking water in India, it was found that the This article is a PNAS Direct Submission. Bihari population was at risk from arsenic exposure due to Freely available online through the PNAS open access option. contamination from naturally occurring trivalent arsenic in the 1To whom correspondence should be addressed. E-mail: [email protected].

www.pnas.org/cgi/doi/10.1073/pnas.1311535110 PNAS Early Edition | 1of6 Downloaded by guest on September 27, 2021 Fig. 1. Arsenic levels in organs of BALB/c mice. At day 28 (A) and day 56 (B), three mice from each group (clear bars, 0 ppm; gray bars, 10 ppm; and black bars, 100 ppm) were culled, and liver, spleen, bone, and kidneys were harvested, homogenized, and digested before analysis of their total arsenic content by ICP- MS (39). In the case of spleen, kidney, and bone of the D28 control samples, these are the weighted mean of two independent analyses. Values are mean ± SE in mg per kg wet weight. *P < 0.05 compared with 0-ppm group.

of arsenic in the organs in which they reside. This was assessed parasite did not affect the metabolism of arsenic. Likewise, the through exposing uninfected BALB/c mice to arsenic at 0, 10, and level of residual total antimony in livers of infected mice 72 h 100 ppm for a total of 56 d and measuring arsenic accumulation in after the end of treatment with Pentostam was not significantly organs and other tissues by ICP-MS. The mice in the 10-ppm altered between non–arsenic-exposed and arsenic-exposed groups group, which is equivalent to tube well arsenic concentrations (Table 1), suggesting that exposure to arsenic does not interfere found in Bihar, gained weight in a similar fashion to the control fl · −1 with the accumulation of antimony in the infected tissues. group at 0 ppm and had an average uid intake of 2 mL d The amounts of infected spleen from the 0-ppm group available (equivalent to 20 μg arsenic per mouse per day). However, mice in for arsenic analysis (Table 1) were limited as a result of the need to the 100-ppm group gained weight at a slower rate, and their fluid − intakes progressively decreased from an average of 1 mL·d 1 to 0.5 isolate parasites for further passage into mice and for drug sensi- − mL·d 1 (equivalent to 50–100 μg arsenic per mouse per day), in- tivity assays. The high SE for the spleen samples from the control dicating cumulative toxicity at the higher arsenic concentration. The mice is a result of the low arsenic content and the low sample arsenic content of standard laboratory chow was determined to be amounts resulting in highly variable count rates during ICP-MS − 0.048 ± 0.005 μg·g 1. Food intake was not measured, but based analysis indicating a large relative variability of arsenic in the con- on a daily intake of 16 g per 100 g body weight for BALB/cByJ trol group. An independent measurement of splenic arsenic content mice (11), arsenic intake from food is negligible (0.15 μgper from three infected mice at 28 d was performed using larger splenic mouse per day). samples, and the results are shown in parentheses in Table 1. The tissue levels of arsenic correlated with the concentration The elevated levels of arsenic seen in the liver and kidney ’ of arsenic in the animals drinking water (Fig. 1). In mice exposed would be expected because these organs are the main sites of to 10 ppm arsenic, levels were maintained or increased between arsenic metabolism and excretion, respectively (12). Liver biopsy day 28 (Fig. 1A) and day 56 (Fig. 1B), times corresponding to the of chronically exposed patients in Bangladesh demonstrated high start and end of the experimental infection. However, in the 100- ppm group, the mean arsenic content of all tissues except the levels of arsenic, between 0.5 and 6 mg per kg dry weight (13). spleen decreased, most likely as a result of the decreased fluid Levels between 0.4 and 1.5 mg per kg wet weight were seen in the intake in the 100-ppm group. livers of BALB/c mice exposed to arsenic at 100 ppm throughout The mean arsenic contents of infected liver and spleens iso- the arsenic exposure time course (days 7, 14, 28, and 56). These lated on day 56 were comparable to those from uninfected ani- experimental results are comparable to those found with similar mals (Table 1), suggesting that the presence of the Leishmania exposures in C3H mice in the literature (14, 15).

Table 1. Total arsenic and antimony in organs of BALB/c mice exposed to arsenic in drinking water − − Total arsenic, mg·kg 1 Total antimony, mg·kg 1

Organ 0 ppm 10 ppm 100 ppm 0 ppm 10 ppm 100 ppm

Liver Infected 0.028 ± 0.011 0.12 ± 0.05 0.41 ± 0.09*** 2.3 ± 0.1 2.2 ± 0.4 2.0 ± 0.1 Uninfected 0.0020 ± 0.0006 0.12 ± 0.02** 0.53 ± 0.14** ND ND ND Spleen Infected 0.12 ± 0.08 (0.01 ± 0.0004)† 0.23 ± 0.18 0.35 ± 0.28 2.4 ± 0.2 2.6 ± 0.1 1.1 ± 0.2* Uninfected 0.0026 ± 0.0007 0.12 ± 0.02** 0.40 ± 0.12* ND ND ND

Arsenic levels were measured by ICP-MS in the livers and spleens of mice that have been exposed to 0, 10, and 100 ppm for 56–63 d. The results represent the mean and SE of measurements from three infected mice at passages 2, 3, 4, and 5 for liver and at passages 3 and 5 for spleen and from uninfected mice at 56 d. Total antimony results are mean and SE of measurements from three mice from − each arsenic exposure level at ﬁfth passage, 72 h post-s.c. injection of 50 mg·kg 1 of Pentostam. ND, not determined. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with control. †Results of an independent experiment.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1311535110 Perry et al. Downloaded by guest on September 27, 2021 Generation of a Pentostam-Resistant Leishmania Line Through in Vivo Exposure to Arsenic. LV9 parasites which were exposed to arsenic in murine hosts in serial passages (Fig. 2) developed resistance to antimonial preparations when assessed in vitro in macrophages (Fig. 3). The level of ex vivo amastigote sensitivity to Pentostam in the in-macrophage assay gradually decreased at each passage. After the third passage the EC50 for parasites exposed at 10 ppm was ﬁvefold greater than the 0-ppm control group (0 ppm, 43 ± 6 − − μg·mL 1; 10 ppm, 228 ± 75 μg·mL 1), and no response to Pentostam − was seen at 500 μg·mL 1 for 100 ppm-exposed parasites. After the −1 ﬁfth passage, the 0-ppm mice had an EC50 of 38.5 ± 3.9 μg·mL , − andnoresponsetoPentostamat500μg·mL 1 was observed in either of the arsenic-exposed parasite groups (Fig. 3). Following passage in mice for a further 4 mo without arsenic exposure, com- − plete resistance to Pentostam at 500 μg·mL 1 was retained, whereas −1 the control group had an EC50 of 60.6 ± 13 μg·mL .

In Vivo Assessment of Leishmania Resistance. On the fifth passage, BALB/c mice exposed to arsenic at 0, 10, and 100 ppm were treated in vivo with drug vehicle, Pentostam, or miltefosine. fi P < Fig. 3. Susceptibility of in-macrophage ex vivo amastigotes to Pentostam. There was a signi cant difference in response ( 0.05) to Ex vivo amastigotes recovered from BALB/c mice exposed to 10 ppm (■)and Pentostam treatment with less than 20% suppression of parasite 100 ppm (▲) arsenic were assessed for their sensitivity to Pentostam for 72 h load in the arsenic-exposed groups compared with 66.4% sup- within mouse peritoneal macrophages (38). Amastigotes from the 0-ppm (●) ± μ · −1 pression in the 0-ppm control group (Fig. 4). In Fig. 4A, milte- BALB/c mice had an EC50 value for Pentostam of 38.5 3.9 g mL , whereas − fosine at 12 mg·kg 1 had a similar effect on the parasite burden parasites recovered from the 100-ppm group of mice remained insensitive to −1 in all three groups. When the data are presented as a percentage the drug at concentrations up to 500 μg·mL . of the control in Fig. 4B, there is a significant difference between

the response of 0-ppm and 10-ppm groups, which may be due to MICROBIOLOGY efficacy is less than 50% (5), and it is the only region where arsenic variability in parasite burden in the control groups. The lack of contamination of the groundwater and VL coexist (18). efficacy of Pentostam in vivo in the arsenic-exposed groups Arsenic has long been known to induce cross-resistance to an- confirms that the cross-resistance developed by the LV9 para- timony in vitro (10), but this phenomenon has not been demon- sites is relevant in vivo as well as in vitro. strated in vivo. This study shows that the speed of resistance Discussion development is related to the levels of arsenic exposure: at high −1 fi levels of hepatic arsenic of between 0.4 and 1.5 mg·kg seen in the The isolated development of decreased antimonial ef cacy in L. donovani Bihar, India, when the drug remains efficacious worldwide in other 100-ppm group, resistance in parasites developed VL-endemic communities has previously been attributed to erratic relatively rapidly over a few months, whereas resistance took prescribing practices and India’s unregulated private healthcare longer to develop at the lower level of exposure of 10 ppm. This latter level, being equivalent to a 60-kg person drinking 3 L of system (16, 17). This study demonstrates, through an in vivo model, μ · −1 that it is biochemically and physiologically plausible that the water contaminated with arsenic at 1,200 g L (19), is in the transmission of L. donovani through arsenic-exposed populations upper bracket of arsenic concentrations found in Bihar (8). has contributed to the development of antimonial-resistant para- The limitations of this work concern how far the models we sites. The Indian subcontinent is the only region where antimonial have used can relate to the development of antimonial resistance in Leishmania in patients in Bihar. Our model of arsenic exposure used high concentrations that recreated in months a physiological situation that would have developed in arsenic-exposed persons over years, as confirmed by the ICP-MS measurements. Thus, even though 10 ppm is equivalent to a high level of arsenic contamination, it is possible that through accumulation, these hepatic arsenic levels can be reached over time at lower exposure levels in mice. Furthermore, the incubation period of VL in humans ranges from weeks to many months (1); therefore, it is possible that at these and lower levels of hepatic arsenic exposure, resistance in the Leishmania parasite may have a chance to develop. There was no significant difference seen in antimony levels between arsenic-exposed and non–arsenic-exposed post- Pentostam treatment livers, indicating that competing metabolism of the two metalloids is unlikely to be a contributory factor in the decreased efficacy of Pentostam in vivo. The stability of drug resistance following passage of resistant lines through non–arsenic-exposed mice implies that the mechanisms of resistance have been acquired at a genetic rather than at an epigenetic level within the parasite. This stability, together with an increased fitness, has been demonstrated in field isolates (20). In relation to this hypothesis, this allows for dissemination of resistant strains through non–arsenic-exposed persons and Fig. 2. Procedure for generation of an antimonial-resistant line in vivo into areas where there is no arsenic contamination through through oral exposure to arsenic. human migration.

Perry et al. PNAS Early Edition | 3of6 Downloaded by guest on September 27, 2021 Fig. 4. Effect of drug treatment on the parasite burden of arsenic-exposed BALB/c mice. Parasite burden is expressed as total parasite numbers per liver (A) and response to treatment as a percentage of the respective untreated control group (B). Following 28 d of infection, at each arsenic exposure level group (clear bars, 0 ppm; gray bars, 10 ppm; and black bars, 100 ppm), BALB/c mice were treated daily for 5 d with either s.c. injections of drug vehicle alone (n = 4, 5, − − and 5 at each arsenic exposure level), 50 mg·kg 1 Pentostam s.c. (n = 3, 5, and 5), or miltefosine 12 mg·kg 1 (n = 3, 4, and 4). Values are mean ± SE. *P < 0.05 compared with 0-ppm group.

Resistance in L. donovani has been studied for over 30 y using to antimony was not determined, but a related study found no both laboratory-generated resistant strains and clinical isolates. correlation between parasite resistance and clinical outcome to These differing methods have led to varying reports of resistance antimonial therapy (35). Detailed epidemiological work is un- mechanisms (16, 21). The main mechanisms identified relate to derway to explore the relationship between arsenic exposure and the parasites increasing their defense against oxidative stress by antimonial treatment failure in VL in Bihar, including assess- manipulating their thiol metabolism and changes in transport of ment of established clinical risk factors. both drug and thiols (16, 22–25). Although ex vivo Pentostam- This work highlights the need to consider the environmental sensitive and -resistant parasites are able to infect mouse perito- context in which a parasite is propagating. There are parallels with neal macrophages, we have been unable to transform these par- development of chloroquine resistance in the malarial parasite asites into insect-stage promastiogtes for biochemical studies. following the public health program of mass administration of The in-macrophage assay system is well established as an assay chloroquine medicated salt in the 1950s and 1960s in South for assessing parasite susceptibility but has been criticized for America, Southeast Asia, and Africa (36). The presence of poor nonstandardization across research groups (26) and not being prescribing practice in Bihar and the coexistence of arsenic reflective of the clinical prognosis (27). In the case of antimonial contamination of the groundwater in this region provide multiple drugs specifically, this lack of clinical correlation may be due to ways for the parasite to be exposed to subtherapeutic doses of the dual mechanism of action of Pentostam, which exerts its Leishmania the closely related metalloids leading to resistance. We conclude direct toxic effects on the parasite not only after fi reduction to SbIII but also via indirect effects on the host immune that arsenic contamination may have played a signi cant role in system leading to increased oxidative and nitrosative stress (28). Bihar because issues of inadequate treatment courses are not fi Therefore, the results of the in vivo treatment with Pentostam exclusive to India, but low antimonial ef cacy is. fi provide important con rmation that the resistance developed Materials and Methods by the arsenic-exposed Leishmania parasites is relevant within Ethics Statement. All animal experiments were approved by the Ethical Review a physiological system. There is some evidence that chronic ar- Committee at the University of Dundee and performed under the Animals senic exposure leads to immunosuppression (29, 30). This could fi fi fi (Scienti c Procedures) Act 1986 (UK Home Of ce Project License PPL 60/4039) in have contributed to the decreased ef cacy of in vivo antimonials in accordance with the European Communities Council Directive (86/609/EEC). our model because part of their mode of action is driven by the immune system (28). However, the current literature is sporadic Animals. BALB/c mice (commercially acquired from Harlan) were used for all in and can be contradictory (31, 32), so no concrete conclusions can vivo experiments. be drawn without further work in this area. Moreover, it cannot explain the parasite resistance observed in vitro in macrophages. Cell Lines. L. donovani (LV9 strain; World Health Organization designation Outside of Bihar, elevated rates of treatment failure for pen- MHOM/ET/67/HU3) ex vivo amastigotes derived from hamster spleens, as tavalent antimonials have been reported from Nepal and Peru previously described (37), were used to infect BALB/c mice by i.p. injection of (33, 34). In Nepal the overall treatment failure rate with sodium 1 × 107 amastigotes (200 μL). Amastigotes were then propagated through stibogluconate was ∼1 in 10. This is considerably lower than BALB/c mice, with infected spleens harvested at 1–2 monthly intervals and failure rates in Bihar which fall below 50% (5), and one of the the recovered parasites used to infect further groups of mice. These para- significant risk factors for treatment failure identified in this sites were propagated in this manner for 1 y before use in the creation of an Nepalese study was living in a district that borders Bihar. Other in vivo resistant line. important clinical risk factors were prolonged course of infection Chemicals. All chemicals used are from Sigma Aldrich unless otherwise stated: and differences in treatment administration. In Peru, a study on AsIII (as sodium meta arsenite) and SbV (as Pentostam, sodium stibogluconate the use of pentavalent antimonials in cutaneous leishmaniasis free of m-chlorocresol; a gift from GlaxoSmithKline). reported a 24.4% failure rate. Patients who experienced treatment failure in Peru were more likely to have had a short disease In Vitro in-Macrophage Drug Sensitivity Assay. Sensitivity of the BALB/c ex vivo Leishmania duration, have more than one CL lesion, have either amastigotes in macrophage was assessed, as described previously (38). EC50 peruviana or Leishmania braziliensis isolated, and have lived in an values were calculated using a two-parameter equation in GraFit (version area of endemicity for less than 72 mo (34). In vitro susceptibility 5.0.13; Erithacus Software):

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1311535110 Perry et al. Downloaded by guest on September 27, 2021 % material (Seronorm Trace Elements Urine; Seronorm) were 197 ± 5 μg As per = 100 ! : y s L(n = 3, certiﬁed value 184 ± 17 μg As per L), and those measured in TORT-2 x 1 + (Lobster Hepatopancreas; National Research Council Canada; http://archive. IC50 nrc-cnrc.gc.ca/obj/inms-ienm/doc/crm-mrc/eng/TORT-2_e.pdf) were 20 ± 0.4 mg As per kg dry weight (certiﬁed 21.6 ± 1.8 mg As per kg dry weight).

Oral Exposure of BALB/c Mice to AsIII in Drinking Water. In designing the arsenic In Vivo Selection of Pentostam Resistance. BALB/c mice, aged between 6 and exposure model, the concentrations of arsenic to be administered to mice 10 wk, were divided into three groups receiving AsIII at 0, 10, or 100 ppm in were chosen to recreate two situations reported from the Indian sub- their drinking water. The mice were preexposed to arsenic for 1 mo before continent. Using an established dose-equivalent equation based on body infection by i.p. injection of 0.2 mL of 1 × 107 L. donovani ex vivo splenic surface area (19), BALB/c mice drinking water containing AsIII at 10,000 − amastigotes from BALB/c mice, as described above. At 28-d postinfection, μg·L 1 (hereafter 10 ppm, equivalent to 133 μM) is equivalent to a 60-kg man − − the mice were euthanized, and ex vivo amastigotes from the spleens of each drinking 3 L·d 1 (18) of water contaminated with arsenic at 1,200 μg·L 1. This group were prepared separately. These amastigotes were either used to concentration has been reported in arsenic-contaminated tube wells in Bihar −1 reinfect further arsenic preexposed groups of mice or, using the in-macro- (8). The higher level of 100,000 μg·L (hereafter 100 ppm, equivalent to 1.33 mM) is equivalent to concentrations greater than those reported from Bihar phage assay described above, assessed for their sensitivity to Pentostam. fl but was chosen to recreate arsenic levels previously detected on liver biopsy See Fig. 1 for an experimental ow diagram. Samples of Leishmania-infected in patients chronically exposed to arsenic (13). liver and spleen were also prepared and analyzed for arsenic content by fi BALB/c mice, aged between 6 and 10 wk, were divided into three groups ICP-MS, as above. Following the fth passage, ex vivo amastigotes were that received either normal tap water (hereafter 0 ppm), arsenic at 10 ppm used to infect groups of mice that had not been exposed to arsenic to look − − (10 mg·L 1), or arsenic at 100 ppm (100 mg·L 1). The water of both groups for stability of resistance. These parasites were passaged in BALB/c mice was changed one to two times per week, water intake was measured, and for 4 mo before reassessment of their sensitivity to Pentostam using the the animals were weighed weekly. All mice were fed standard autoclavable in-macrophage assay. murine chow pellets RM1 A (Special Diets Services). At days 7, 14, 28, and 56, three mice from each exposure level were eu- In Vivo Assessment of Parasite Resistance. In vivo sensitivity to Pentostam and thanized. Harvested liver and kidney were placed in CK14 tubes containing miltefosine was assessed at the end of the fifth passage. At 28 d postinfection, ceramic beads, and harvested spleen was placed in MK28R tubes containing groups of mice from each exposure level were treated with drug vehicle only −1 metal beads (PeqlabBiotechnologie GmbH) with 500 μL double-distilled (sterile water, s.c. injection), Pentostam (50 mg·kg , s.c.), or miltefosine (12 −1 water and homogenized at 2 × 5,000 rpm for 10 s with a 20-s gap, using mg·kg , orally) once daily for 5 d. Fresh drug dosing solutions were pre- a PreCelleys 24 homogenizer (Bertin Technologies). Harvested bone was pared daily. On day 35 postinfection, 72 h after the final treatment, all crushed with a pestle and mortar before mechanical homogenization, in animals were euthanized, and parasite burdens were determined by blinded MK28R tubes, at 2 × 5,900 rpm for 30 s with a 15-s gap. Blood cells and sera counting. The total number of parasites per liver was calculated by multi- MICROBIOLOGY were separated by centrifugation at 15,000 × g for 90 s. Samples of murine plying the parasites per organ cell nucleus by 2 × 105 by the weight of the chow were also collected for digestion and analysis. liver in mg (40). The differences in response to Pentostam and miltefosine Samples (100-mg wet weight) were digested in closed microwave vessels between arsenic exposure groups were compared using the unpaired Stu- using 500 μL nitric acid and 100 μL hydrogen peroxide followed by micro- dent t test. Liver and spleens from treated animals were prepared as de- wave digestion, further dilution with double-distilled water, and analysis by scribed above and analyzed for both arsenic and antimony by ICP-MS. ICP-MS (39). Each mouse tissue sample was measured in triplicate and expressed as mg per kg wet weight. The average limit of detection was ACKNOWLEDGMENTS. This work was supported by the Wellcome Trust Grants 0.2 μg As per kg wet weight. Arsenic values measured in certified reference 090665 (to M.R.P.) and 079838 and 083481 (to A.H.F.).

1. Herwaldt BL (1999) Leishmaniasis. Lancet 354(9185):1191–1199. 19. Reagan-Shaw S, Nihal M, Ahmad N (2008) Dose translation from animal to human 2. Guerin PJ, et al. (2002) Visceral leishmaniasis: Current status of control, diagnosis, and studies revisited. FASEB J 22(3):659–661. treatment, and a proposed research and development agenda. Lancet Infect Dis 2(8): 20. Vanaerschot M, et al. (2011) Antimonial resistance in Leishmania donovani is associ- 494–501. ated with increased in vivo parasite burden. PLoS ONE 6(8):e23120. 3. Chappuis F, et al. (2007) Visceral leishmaniasis: What are the needs for diagnosis, 21. Singh N, Singh RT, Sundar S (2003) Novel mechanism of drug resistance in kala azar treatment and control? Nat Rev Microbiol 5(11):873–882. field isolates. J Infect Dis 188(4):600–607. 4. Singh RK, Pandey HP, Sundar S (2006) Visceral leishmaniasis (kala-azar): Challenges 22. Borst P, Ouellette M (1995) New mechanisms of drug resistance in parasitic protozoa. ahead. Indian J Med Res 123(3):331–344. Annu Rev Microbiol 49:427–460. 5. Olliaro PL, et al. (2005) Treatment options for visceral leishmaniasis: A systematic 23. Ouellette M, et al. (1995) Transport of antifolates and antimonials in drug-resistant – review of clinical studies done in India, 1980-2004. Lancet Infect Dis 5(12):763 774. Leishmania. Drug Transport in Antimicrobial and Anticancer Chemotherapy, Infectious 6. Matlashewski G, et al. (2011) Visceral leishmaniasis: Elimination with existing inter- Disease and Therapy, eds Scully BE, Neu HC (Marcel Dekker, New York), Vol 17, pp 377–402. – ventions. Lancet Infect Dis 11(4):322 325. 24. Ashutosh SS, Sundar S, Goyal N (2007) Molecular mechanisms of antimony resistance 7. Perry MR, et al. (2011) Visceral leishmaniasis and arsenic: An ancient poison contributing to in Leishmania. J Med Microbiol 56(Pt 2):143–153. antimonial treatment failure in the Indian subcontinent? PLoS Negl Trop Dis 5(9):e1227. 25. Aït-Oudhia K, Gazanion E, Vergnes B, Oury B, Sereno D (2011) Leishmania antimony re- 8. Chakraborti D, et al. (2003) Arsenic groundwater contamination in Middle Ganga sistance: What we know what we can learn from the field. Parasitol Res 109(5):1225–1232. Plain, Bihar, India: A future danger? Environ Health Perspect 111(9):1194–1201. 26. Seifert K, Escobar P, Croft SL (2010) In vitro activity of anti-leishmanial drugs against 9. Yan S, Jin L, Sun H (2005) Sb Antimony in Medicine. Metallotherapeutic Drugs and 51 Leishmania donovani is host cell dependent. J Antimicrob Chemother 65(3):508–511. Metal-Based Diagnostic Agents—The Use of Metals in Medicine (John Wiley, Chi- 27. Rijal S, et al. (2007) Antimonial treatment of visceral leishmaniasis: Are current in vitro chester, UK), pp 441–461. susceptibility assays adequate for prognosis of in vivo therapy outcome? Microbes 10. Dey S, et al. (1994) High level arsenite resistance in Leishmania tarentolae is mediated Infect 9(4):529–535. by an active extrusion system. Mol Biochem Parasitol 67(1):49–57. 28. Rais S, et al. (2000) Sodium stibogluconate (Pentostam) potentiates oxidant production 11. Bachmanov AA, Reed DR, Beauchamp GK, Tordoff MG (2002) Food intake, water intake, in murine visceral leishmaniasis and in human blood. Antimicrob Agents Chemother and drinking spout side preference of 28 mouse strains. Behav Genet 32(6):435–443. – 12. Vahter M, Concha G (2001) Role of metabolism in arsenic toxicity. Pharmacol Toxicol 44(9):2406 2410. 29. Biswas R, et al. (2008) Analysis of T-cell proliferation and cytokine secretion in the 89(1):1–5. – 13. Mazumder DNG (2005) Effect of chronic intake of arsenic-contaminated water on individuals exposed to arsenic. Hum Exp Toxicol 27(5):381 386. liver. Toxicol Appl Pharmacol 206(2):169–175. 30. Bishayi B, Sengupta M (2003) Intracellular survival of Staphylococcus aureus due to 14. Kitchin KT, Conolly R (2010) Arsenic-induced carcinogenesis—Oxidative stress as alteration of cellular activity in arsenic and lead intoxicated mature Swiss albino mice. – a possible mode of action and future research needs for more biologically based risk Toxicology 184(1):31 39. assessment. Chem Res Toxicol 23(2):327–335. 31. Soto-Peña GA, et al. (2006) Assessment of lymphocyte subpopulations and cytokine 15. Ahlborn GJ, et al. (2009) Impact of life stage and duration of exposure on arsenic- secretion in children exposed to arsenic. FASEB J 20(6):779–781. induced proliferative lesions and neoplasia in C3H mice. Toxicology 262(2):106–113. 32. Wu MM, Chiou HY, Ho IC, Chen CJ, Lee TC (2003) Gene expression of inflammatory 16. Croft SL, Sundar S, Fairlamb AH (2006) Drug resistance in leishmaniasis. Clin Microbiol molecules in circulating lymphocytes from arsenic-exposed human subjects. Environ Rev 19(1):111–126. Health Perspect 111(11):1429–1438. 17. Hasker E, et al. (2010) Management of visceral leishmaniasis in rural primary health 33. Rijal S, et al. (2010) Clinical risk factors for therapeutic failure in kala-azar patients treated care services in Bihar, India. Trop Med Int Health 15(Suppl 2):55–62. with pentavalent antimonials in Nepal. Trans R Soc Trop Med Hyg 104(3):225–229. 18. Ravenscroft P, Brammer H, Richards K (2009) Arsenic Pollution: A Global Synthesis 34. Llanos-Cuentas A, et al. (2008) Clinical and parasite species risk factors for pentavalent an- (Wiley-Blackwell, Chichester, UK). timonial treatment failure in cutaneous leishmaniasis in Peru. Clin Infect Dis 46(2):223–231.

Perry et al. PNAS Early Edition | 5of6 Downloaded by guest on September 27, 2021 35. Yardley V, et al. (2006) American tegumentary leishmaniasis: Is antimonial treatment 38. Wyllie S et al. (2012) The anti-trypanosome drug fexinidazole shows potential for outcome related to parasite drug susceptibility? J Infect Dis 194(8):1168–1175. treating visceral leishmaniasis. Sci Transl Med 4(119):119re1. 36. Payne D (1988) Did medicated salt hasten the spread of chloroquine resistance in 39. Williams PN, et al. (2007) Greatly enhanced arsenic shoot assimilation in rice leads to el- Plasmodium falciparum? Parasitol Today 4(4):112–115. evated grain levels compared to wheat and barley. Environ Sci Technol 41(19):6854–6859. 37. Wyllie S, Fairlamb AH (2006) Reﬁnement of techniques for the propagation of 40. Stauber LA (1958) Host resistance to the Khartoum strain of Leishmania donovani. Leishmania donovani in hamsters. Acta Trop 97(3):364–369. Rice Inst Pam 45(1):80–96.

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