ELSEVIER Molecular and Biochemical Parasitology 66 (1994) 161-164

MOLECULAR AND BIOCHEMICAL PARASITOLOGY

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Atomic absorption spectrophotometric measurement of intracellular arsenite in arsenite-resistant Leishmania

Ajay Kumar Singh, Hsing Yin Liu, Sho Tone Lee *

Division of lnfectious Diseases, Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan

Received 16 February 1994; accepted 9 May 1994

Key words: Leishmania; DNA amplification; Drug resistance

Leishmania spp. made resistant to methotrexate (MTX) have been found to be cross-resistant to sodium arsenite (Ars) and vice versa [1,2]. The ac- quired resistance has been attributed to the presence of amplified H circle DNA [1,3,4]. It is clear now that the cross resistance observed is due to the presence of two separate genes in some of the H circles. By mapping H circles of various lengths from different arsenite-resistant variants [2] and by cloning and transfection studies of DNA fragments derived from H circles [3,5,6], the gene responsible for MTX resistance has been identified as a short chain dehydrogenase [3] or an aldoketo reductase [5,6]. These genes differ from the classical MTX-re- sistant dihydrofolate reductase gene (DHFR) previ- ously described [7]. The gene responsible for Ars resistance is a member of the multidrug resistance (mdr) gene family, but is functionally distinct from the classical mdr gene in mammalian systems [8]. The transport of MTX in variants carrying H DNA

Abbreviations: MTX, methotrexate; Ars, sodium arsenite; DHFR, dihydrofolate reductase; mdr, multidrug resistance.

* Corresponding author. Tel: 886-2-789-9170; Fax: 886-2-785- 3569.

has been well characterized [4], but the influx and efflux of arsenite in these variants has not been examined owing to the lack of suitable arsenite isotopes. In this report, we describe the use of atomic absorption spectrophotometry to study the uptake and release of Ars in Ars-resistant Leishmania mexi- cana amazonensis carrying amplified H-DNA.

Cloned (2-23A) and uncloned (A) variants se- lected for sodium arsenite resistance and having extrachromosomal DNA amplification (69 kb circu- lar DNA) have been described previously [9,10]. The amplified circular DNAs from cloned (2-23A) and uncloned (A) arsenite-resistant variants have been identified as H circle in nature by the specific probe priM 3 (a gift from S.M. Beverley, Harvard Medical School). These arsenite-resistant variants are cross- resistant to MTX (12X) but sensitive to heavy metals like cadmium (CdC12) and lead (PbCI 2) (data not shown). They were also sensitive to hydrophobic drugs such as puromycin and vinblastine (data not shown). The variants possess an mdr-like gene LtpgpA (from L. tarentolae) on their H circle (Fig. la), as determined by the LtpgpA-specific probe PM7 [11] (a gift from M. Ouellette, Laval Univer- sity, Canada). A transcript of approximately 5-5.5 kb was also identified in the total RNA of 2-23A

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162 A.K. Singh et al. / Molecular and Biochemical Parasitology 66 (1994) 161-164

and A variants by the same probe (Fig. lb). No such transcripts were found in wild-type (W) or arsenite- resistant variants without DNA amplification (P~ and 2-23A~) or in a tunicamycin-resistant variant with DNA amplification (T). These data provide evidence that an LtpgpA mdr-like gene and its specific tran- scripts are present in Leishmania mexicana amazo-

nensis made resistant to arsenite and having H-DNA amplification, just as in other Leishmania species [2-4].

Fig. 2 shows the intracellular uptake and release of sodium arsenite by 2-23A (Fig. 2a) when com- pared to its wild-type counterpart (2-23W) and the variant without DNA amplification (2-23A~) as mea- sured by atomic absorption spectrophotometry in a 1-h assay. The wild-type (2-23W) and the 2-23P~ parasites showed increasing accumulation of arsenite with time, while the 2-23A showed consistently lower uptake of the compound throughout the assay period. When all these parasites were washed 1 h after exposure to arsenite and resuspended in arsen- ite-free Hanks' balanced salt solution (HBSS, Gibco) for a release assay (Fig. 2b), the arsenite-resistant 2-23A variants showed rapid loss of about 70-80% of their intracellular arsenite within 5 min of the assay period whereas the wild-type cells and the 2 - 2 3 ~ variants retained almost 70% to 80% of their original arsenite during this period. The quick release of arsenite was not reversed by verapamil (2-23 AV, Fig. 2b) at doses of 10 /xM or 25 /zM. Energy deprivation by culturing the cells in phosphate- buffered saline (1 × PBS) for 4 h at 27°C before the uptake assay led to increasing accumulation of Ars in 2-23A variants to the level in 2-23/~ variants, and to blocking of subsequent release of the drug from the parasites. This may indicate release of drug in this variant is energy dependent. The uncloned wild type (W), A~ and A variants behaved the same as their cloned counterparts (data not shown).

In summary, arsenite-resistant variants of Leish- mania carrying the mdr-like gene in their amplified H-DNA at first glance behave just like mammalim cells carrying the classical mdr gene by releasing arsenite quickly, as measured by atomic absorption spectrophotometry. However, the sensitivity of the resistant cells carrying H-DNA to puromycin and vinblastine and the inability of verapamil to reverse the release argue against the mdr-like gene being a

classical mdr gene. The finding that this gene shows substrate specificity to arsenite but not to other heavy metals such as cadmium and lead implies that it

a b

1 2 3 ~ '~ " < ~- o,

kb kb

23.1-- 9.4--

9.5-- 5.6--

7 .5 - 4.3--

4 4 - -

2.2~

2 .0 - ! 2.4-

I

0.55-- 1.3--

Fig. 1. Presence of LtpgpA mdr-like gene in amplified extrachro- mosomal H-DNA and the existence of its transcripts in L. m. amazonensis resistant to sodium arsenite. (a) Amplified extrachro- mosomal DNA from cloned 2 -23A variants was isolated by alkaline lysis method [14], restricted with BamHl (1), PstI (2) and HaellI (3), electrophoresed in 0.7% agarose and blotted on Nytran membrane (Schleicher and Schuell). The blot was probed with PM7 in Southern hybridization. (b) Total RNA was isolated by a single-step method using guanidinum isothiocyanate/phe- nol/chloroform extraction [15] from W, A, A¢, T, 2 -23A and 2-23A~ variants and 20 /xg RNA from each sample were elec- trophoresed on 1% agarose containing formamide [16]. The blot was also hybridized to PM7 probe. Blots for Southern and North- ern were prehybridized and hybridized as described [16]. PM7 probes were labeled with 32p by multiprime labeling system (Amersham). Blots were washed at RT with 2 X SSC/0.1% SDS twice followed by 0.1 ×SSC/0 .1% SDS twice at 60°C. Exposure to Kodak X-Omat film was carried out for 8 -24 h at - 7 0 ° C [9,10] with the use of an intensifying screen (Dupont). W = uncloned wild type; ~ (uncloned), 2-23P~ (cloned): arsenite-re- sistant variant without DNA amplification; A (uncloned), 2 -23A (cloned): arsenite-resistant variant with DNA amplification; T = tunicamycin resistant variant with DNA amplification; PM7 = a 850 bp PstI insert of H-DNA from L. tarentolae cloned in pGEM-3, specific for LtpgpA gene [11].

A.K. Singh et al. / Molecular and Biochemical Parasitology 66 (1994) 161-164 163

b a

2 0 lOO

1~ r \ , l

.4--"

" i 2--23A' "0 1( t -

" " 4 0

Ol ~ 1 ~ 1 , . . ~ 2-23A I V 2 0

1~ 7o 3"0 4'0 ;o 6'o 5 lOlS 30

Time in min > Fig. 2. Determination of the uptake and release of sodium arsenite in arsenite-resistant variants by atomic absorption spectrophotometry. (a) Arsenite-resistant variants with DNA amplification (2-23A) were cultured in arsenite-free medium for 24 h before use. Parasites were then washed 3 times in arsenite-free Hanks' balanced salt solution (HBSS, Gibco) containing 1 mM EDTA and resuspended in the same solution at 5 × 106 m1-1 containing 200 ~ M arsenite. From time to time after the exposure, 2 × 106 cells were pelleted by centrifugation (Eppendorf, Beckman). The cell pellet was digested with 0.4 ml nitric acid for 3 h and then diluted with 10 ml distilled water. The clear supernatant after centrifugation was incubated with 0.4 ml of concentrated HCI and 3 ml of 20% KI solution for at least 5 min. Total intracellular arsenite concentration was measured by an atomic absorption spectrophotometer [17] (Hitachi Z-8000, Tokyo, Japan) equipped with a hydride formation system (Hitachi-2, Tokyo, Japan) [18]. Wild-type (2-23W) and 2-23,~ variants treated in the same way served as controls. The amount of intracellular arsenite was expressed as ng 10 -6 cells. (b) The same set of parasites (2-23W, 2-23,~ and 2-23A) incubated with sodium arsenite for 1 h as described in 2a were washed and resuspended in arsenite-free medium. From time to time cells were pelleted by centrifugation and treated as described before for determination of intracellular arsenite. Each point represents the average of 3 experiments + S.D. See legend to Fig. 1 for 2-23,~ and 2-23A. 2 -23AV represents the 2 -23A variants treated with 10 p.M verapamil before release assay.

functions more like the heavy metal pumps in prokaryotes [12,13].

Acknowledgements

We thank Dr. Cathy Fletcher for reading the manuscript and Ms. Lola Wen for her secretarial assistance. This work was supported by the National Science Council of the Republic of China (grant NSC 81-0412-B001-04). A.K.S. is Postdoctoral Fel- low, National Science Council, Taiwan, R.O.C.

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