Interaction Arsine

Interaction of Arsine with Hemoglobin in Arsine-Induced Hemolysis

Leonard T. Rael,*,†,1 Felix Ayala-Fierro,‡ Raphael Bar-Or,*,† Dean E. Carter,§ and David S. Barber¶

*Swedish Medical Center, Trauma Research Laboratory, Englewood, Colorado 80113; †DMI BioSciences, Inc., Englewood, Colorado 80113;

‡The Dial Corporation, Product Safety, Regulatory and Microbiology–Clinical Studies and Toxicology, Scottsdale, Arizona 85254; §Department of

Pharmacology and Toxicology, The Center for Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona 85721; and ¶Department of

Physiological Sciences, Center for Environmental and Human Toxicology, University of Florida, Gainesville, Florida 32611

Received June 22, 2005; accepted November 22, 2005

The mechanism of arsine (AsH3) toxicity is not completely

understood, but hemoglobin (Hb) has long been recognized as

a necessary component of the overall mechanism of AsH3-induced

hemolysis. In this study, the role of Hb in AsH3-induced hemolysis

was investigated. The purpose was to determine whether exposure

to AsH3 altered the structure of the heme or globin constituents of

Hb. Arsine was incubated with isolated, human oxyhemoglobin

(oxyHb) and carboxyhemoglobin (carboxyHb), and the release of

heme and formation of AsH3-induced hemoglobin modifications

were examined. Arsine increased the amount of heme released

from oxyHb by 18%. When carboxyHb was incubated with AsH3,

there was no change in heme release, suggesting that the sixth

ligand position on the heme iron may be critical in the interaction

with AsH3. Arsine–Hb interactions were studied by mass spectral

analysis of heme, a-chain globin, and b-chain globin. Arsine had

no significant effect on the a- or b-chain LCMS spectra in oxyHb

and carboxyHb, but in oxyHb, arsine consistently increased the

frequency of methyl acetate ion fragment (�CH2OOH, m/z = 59)

loss from heme in the matrix-assisted laser desorption/ionization-

mass spectrometry (MALDI-MS) spectra. The formation of Hb–

protein crosslinks was investigated by Western blotting using an

anti-Hb antibody in isolated membranes from AsH3-treated

erythrocytes, but no Hb-membrane adducts were found. These

results suggest that the interaction between AsH3 and hemoglobin

result in an increase in heme release which may contribute to the

hemolytic mechanism of AsH3.

Key Words: arsine; oxyhemoglobin; carboxyhemoglobin; heme;

hemolysis.

INTRODUCTION

Arsine gas (AsH3), the hydride of arsenic, is the mostacutely toxic form of arsenic (threshold limit value ¼ 50 ppb;

ACGIH 1982).1 Exposure to AsH3 is possible from theaccidental release of the gas during some manufacturingprocesses. AsH3 is extensively used for epitaxial growth ofgallium arsenide and as a dopant for silicon-based electronicdevices in the semiconductor industry. Accidental exposure toAsH3 may also occur from any situation where arsenic-contaminated metals are treated with a strong acid, e.g., metalmining, paint, and herbicides (Buchanan, 1962). The erythro-cyte is the main target of AsH3, with exposure causinghemolysis. In human exposures to toxic levels of AsH3, clinicalexperience is consistent with intravascular hemolysis, anddark-red urine (hemoglobinuria) is usually the first symptom.This is followed by abdominal pain, jaundice, and anemia(Romeo et al., 1997). Splenomegaly has been observed in miceexposed to low levels of AsH3 for 60 days (Hong et al., 1989).Exposure to AsH3 was fatal in up to 25% of the reported humancases (Fowler and Weissberg, 1974).

The mechanism of AsH3 toxicity is not clearly understood.It has been postulated that AsH3 exerts its toxic effects throughoxidative stress depleting reduced glutathione (Pernis andMagistretti, 1960; Blair et al., 1990), but other investigationshave contradicted the importance of GSH (Hatlelid et al.,1995; Winski et al., 1997). It has also been suggested thatAsH3 interacts with important sulfhydryl groups located onthe membrane Naþ,Kþ-ATPase pump inhibiting the pumpand causing cell swelling and lysis (Levinsky et al., 1970).However, the finding that dog erythrocytes are also hemolysedby AsH3 (Hatlelid et al., 1995) makes the Na,K-ATPase pumpan unlikely target of AsH3 because most dog erythrocyteslack this pump. In addition, AsH3 did not significantly alterATP levels or inhibit the ATPase in human erythrocytes(Winski et al., 1997).

1 To whom correspondence should be addressed at Swedish Medical Center,

Trauma Research Laboratory, 501 E. Hampden Ave., Rm. 4–454, Englewood,

CO 80113. Fax: (303) 788-4064. E-mail: [email protected].

1 In 1999, a new AsH3 TLV-TWA of 2 ppb was proposed but was not adopted

in 2000 or 2001. In 2001, ACGIH proposed to lower AsH3 TLV-TWA from 50

to 3 ppb, and to designate AsH3 as an A-1 (Confirmed Human Carcinogen). In

2003, AsH3 was placed on the ACGIH ‘‘under study’’ list. In 2004, AsH3

appeared with an ACGIH proposed (trial value) TLV-TWA of 5 ppb and A4

designation (Not Classifiable as a Human Carcinogen). This proposal remained

in the 2005 ACGIH booklet.

� The Author 2005. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.For Permissions, please email: [email protected]

TOXICOLOGICAL SCIENCES 90(1), 142–148 (2006)

doi:10.1093/toxsci/kfj054

Advance Access publication December 1, 2005

by guest on March 19, 2011

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Oxyhemoglobin (oxyHb) has long been recognized asa necessary component to the overall mechanism of AsH3-induced hemolysis, as conversion of oxyHb to carboxyHbprevents hemolysis in erythrocytes exposed to AsH3 (Raelet al., 2000). Considerable evidence exists indicating thatexposure to AsH3 causes Hb oxidation and denaturationthat may lead to toxicity (Hatlelid et al., 1996; Blair et al.,1990). It has been proposed that heme-containing proteinsactivate AsH3 to a toxic intermediate, leading to AsH3 toxicity(Ayala-Fierro et al., 1999). The release of free heme (ferripro-toporphyrin IX, hemin) from Hb can produce hemolysis bya colloid-osmotic mechanism (Chou and Fitch, 1981). Similareffects are observed in erythrocytes exposed to AsH3, withmassive potassium loss occurring within 5 minutes of expo-sure, followed by hemolysis (Rael et al., 2000). Based on thesimilarity of toxic events, we investigated whether exposure ofHb to AsH3 led to increased release of free heme.

It has also been shown that erythrocytes exposed to AsH3

retain arsenic (As) in a non-dialyzable form (Graham et al.,1946). This had led to the hypothesis that the formation ofan As–Hb complex may occur and be involved in hemolysis(Fowler and Weissberg, 1974). Finally, it is known that oxi-dation of Hb resulting in Hb denaturation and precipitationcan lead to abnormal association of Hb with erythrocytemembrane proteins that increase the fragility of the erythrocytemembrane (Murakami and Mawatari, 2003). In the presentresearch, human Hb and human erythrocytes treated with AsH3

were used as a model system to investigate these three potentialinteractions of AsH3 with Hb.

MATERIALS AND METHODS

Chemicals

Zinc arsenide (99%) and pyridine (ACS grade) were obtained from Aldrich

Chemical Co. (Milwaukee, WI). All other chemicals, including purified human

hemoglobin, rabbit anti-human hemoglobin antibody, and goat anti-rabbit IgG

(alkaline phosphatase conjugate), were all purchased from Sigma Chemical Co.

(St. Louis, MO).

Arsine Generation

Arsine was generated by the method of Hatlelid et al. (1995). Briefly, zinc

arsenide was reacted with 50% sulfuric acid to generate AsH3 gas, which was

bubbled into 0.02 M phosphate buffer or PBS to the desired concentration using

argon as the carrier gas. Arsine concentration was determined by reaction with

0.55% diethyldithiocarbamate in pyridine, followed by spectrophotometric

determination of this product at 510 nm. Caution: AsH3 is a toxic gas and

appropriate precautions should be taken. All procedures should be performed in

an approved fume hood. A saturated potassium permanganate solution trap, in-

line after the aqueous trap, should be used to prevent the release of AsH3 during

its generation.

Generation of Hemoglobin Species

A dialyzed, 0.8 mM solution of human hemoglobin (MW ¼ 64 kDa) in

a 0.02 M phosphate buffer (pH 7.4) was used in all the heme-release studies, as

well as the studies involving interactions between AsH3 and hemoglobin.

Generation of metHb. Methemoglobin was formed by dissolving human

Hb in 0.02 M phosphate buffer and treating the Hb solution with sodium nitrite.

After addition of sodium nitrite, the solution was dialyzed overnight against at

least two changes of buffer at 4�C in 8000 MW cutoff tubing (Spectrum

Medical Industries, Inc., Houston, TX). The purity and concentration of the

metHb solutions were verified by spectrophotometry with wavelength maxima

of 500 and 631 nm (Zijlstra and Buursma, 1987).

Generation of oxyHb. A total of 1 mg of sodium hydrosulfite was added to

6 ml of metHb solution. The solution was placed in a dialysis bag and dialyzed

overnight in phosphate buffer under constant stirring, changing the buffer at

least once. The solution turned red, and oxyHb formation and concentration

were verified by spectrophotometry with wavelength maxima of 542 nm and

577 nm (Zijlstra and Buursma, 1987).

Generation of carboxyHb. CarboxyHb was produced using the oxyHb

solution described above. The oxyHb solution was bubbled with carbon

monoxide gas for approximately 15–20 min. The solution turned a bright red,

indicating the replacement of oxygen with carbon monoxide. CarboxyHb

formation and concentration were verified by spectrophotometry at 539 nm and

569 nm (Zijlstra and Buursma, 1987).

Determination of Heme Release

For these studies, all three Hb species (metHb, oxyHb, and carboxyHb) were

assessed for heme release in the presence of AsH3. For each Hb species, four

separate 0.5 ml incubations were performed in triplicate at room temperature.

Each incubation contained 0.25 ml of a 0.8 mM Hb solution (final ¼ 0.4 mM).

In each specific Hb species, the negative control was prepared by adding

0.25 ml of 0.02 M phosphate buffer to the hemoglobin solution. The positive

control consisted of 2.5 M urea in PBS and was prepared by adding 0.125 ml

of 10 M urea and 0.125 ml phosphate buffer to the 0.8 mM Hb solution. Two

different AsH3-treated samples were prepared. The first AsH3-treated incu-

bation contained Hb and AsH3 only and was prepared by adding 0.125 ml of

1.6 mM AsH3 in phosphate buffer (final AsH3 concentration ¼ 0.4 mM) and

0.125 ml of phosphate buffer to each Hb solution. The second AsH3-treated

incubation contained Hb, AsH3, and urea, and was prepared by adding 0.125 ml

of 10 M urea and 0.125 ml of 1.6 mM AsH3 to each 0.8 mM Hb solution. An

aliquot of the incubation was diluted and scanned on the spectrophotometer to

determine if any changes in the Hb spectra occurred after incubation with AsH3.

The assay for the quantification of free hemin was based on the extraction

procedure given by Letarte et al. (1993). Briefly, after a 30-min incubation,

1 ml of 0.1 M ammonium acetate (pH 4.0) was added to each incubation

and stirred gently for 10 seconds. This process converted the free hemin to

hematin. The samples were then extracted with two consecutive 5-ml washes

with chloroform:methanol (2:1). After each wash, the samples were spun at

2000 rpm for 10 min. After the extraction process, the organic layer (bottom

layer) was collected. The organic extract was then evaporated to dryness in

a water bath with nitrogen gas at 35�–40�C. The dry extract was re-dissolved

in 0.5 ml of DMSO:chloroform (1:4) and stirred vigorously for 30 s. Hematin

was then back-extracted into the aqueous phase using 1 ml of 0.1 N NaOH.

The samples were spun for 10 min at 2000 rpm. The aqueous phase (0.8 ml)

was transferred to a disposable polystyrene cuvette along with 1.6 ml 0.1 N

NaOH. The samples were then read on a Beckman DU-7 spectrophotometer

at 385 nm.

Arsine–Hemoglobin Interactions

The negative control and AsH3-treated incubation mixtures from the heme

release study for all three Hb types were analyzed by mass spectrometry. The

samples were diluted 1:50 in dH2O and analyzed by high performance liquid

chromqtography (HPLC; Waters Corporation, Milford, MA) coupled to

positive electrospray ionization time-of-flight mass spectrometry (þESI-TOF

MS, Micromass, UK). An aliquot of 25 ll of each sample was injected

onto a YMC-Pack Protein-RP, 150 mm 3 4.6 mm, 5l, HPLC column

heated to 50�C (Waters Corporation) using a 20-min linear gradient method

using water/0.1% trifluoroacetic acid (A) and acetonitrile/0.1% TFA (B).

INTERACTION OF ARSINE WITH HEMOGLOBIN 143



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The chromatographic and mass spectrometry conditions were as previously

described (Bar-Or et al., 2005). The mass spectra for the a- and b-chains of

globin were deconvolved to the uncharged parent mass using MaxEnt 1

(Micromass, UK). The same samples were analyzed by matrix-assisted laser

desorption/ionization (MALDI)-qTOF (Micromass, UK) by spotting 1 ll of a

1:50 dilution of the negative control or AsH3-treated incubations mixed 1:1

with 5 lg/ml alpha-Cyano-4-hydroxycinnamic acid. Samples were analyzed in

positive V-optics reflectron mode with a scan range of 300–1000 Da using

a collision energy of 10 eV.

Arsine-Induced Hemoglobin Associations with Membrane Constituents

Blood was collected by venipuncture from healthy male and female

volunteers (ages 22–40 years). The blood was sedimented by centrifugation

(2000 rpm, 10 min) and rinsed twice with glucose-containing phosphate-

buffered saline (glucose-PBS, pH 7.4) in order to remove plasma and buffy

coat. Packed erythrocytes were treated with PBS (control) or 0.5 mM AsH3

(final) for 5 or 30 min at 37�C. At each time point, a 0.5-ml aliquot was

removed, and the erythrocytes were lysed and then centrifuged at 14,000 rpm,

after which the supernatants were removed. Membranes were washed three

more times and the membrane pellet was white. Total protein content was

determined using the BCA assay (Pierce Biotechnology, Rockford, IL). A total

of 35 lg of membrane protein was separated on a 6% sodium dodecyl sulfate-

polyacrylamide gel electrophoresis (SDS-PAGE) gel and stained with Coomasie

blue. Proteins from a duplicate gel were transferred to a polyvinylidene

difluoride (PVDF) membrane and probed with a rabbit anti-human hemoglobin

antibody (Sigma, St. Louis, MO). An alkaline phosphatase conjugated goat

anti-rabbit IgG secondary antibody (Sigma) was used at a 1:10,000 dilution,

and the blot was developed with BCIP/NBT (Sigma Fast tablets, Sigma).

Data Analysis

All data are expressed as mean ± standard deviation. Values are denoted

with an asterisk (*) if statistically different from controls (p < 0.05), using

Student t-test (Microsoft Excel).

RESULTS

Interaction of Arsine with Isolated Human Hemoglobin

Heme release. Treatment of isolated human Hb with AsH3

increased the amount of heme released from oxyHb by 0.88lM versus controls (Fig. 1A). This increase corresponded to an18.3% increase in the AsH3-treated oxyHb compared tocontrols, whereas an 18.5% (0.9 lM) and 43.6% (2.1 lM)increase were observed in urea-treated and AsH3–urea-treatedoxyHb, respectively. For carboxyHb, there was no significantdifference between AsH3-treated carboxyHb and controls,although the combination of AsH3 and urea caused an increasein heme release, albeit an insignificant one (Fig. 1B). This trendfor carboxyHb was also observed for metHb in that AsH3 hadno significant effect on heme release (data not shown). Spectralanalysis of hemoglobin samples after incubation with AsH3

demonstrated that absorbance spectra were identical to thoseobtained prior to the addition of AsH3 (data not shown).

Direct reaction between AsH3 and hemoglobin. To in-vestigate any possible alterations of the Hb constituents, theincubations from the heme release study were analyzed bymatrix-assisted laser desorption/ionization mass spectrometry

(MALDI-MS) and liquid chromatography–mass spectrometry(LCMS). The MALDI-MS analyses of the heme groups (m/z ¼616) derived from AsH3-treated human hemoglobin showed nopeaks that corresponded to the addition of the arsenic species toheme. However, there was an increase in the abundance ofheme fragments in the AsH3-treated samples of oxyHb (Fig.2A) and metHb (Fig. 2B), but not for carboxyHb (Fig. 2C). Thepeaks at m/z ¼ 557.2 and m/z ¼ 498.2 have been previouslyidentified as the loss of one or two �CH2COOH fragments (–59per fragment) from the native heme molecule (Demirev et al.,2002). The LCMS analyses of the a- and b-chains of the AsH3-treated hemoglobin were virtual identical to control (data notshown). Both treated and control samples showed an a-chainpeak at 15,127 Da, which corresponded to the theoretical value(15,126 Da). Likewise, both samples showed a b-chain peak at15,868 Da, corresponding to the theoretical value of 15,867 Da.No evidence of covalent modification was seen throughout thespectra of the a- or b-chain of globin.

Arsine-induced hemoglobin associations with erythrocytemembrane constituents. No differences in protein abundancewere observed between control and AsH3-treated samples

FIG. 1. Arsine-induced heme release from human hemoglobin. 400 lM

oxyHb (A) or carboxyHb (B) were incubated with phosphate buffer (control),

2.5 M urea, 0.4 mM AsH3, or 0.4 mM AsH3 þ 2.5 M urea for 30 min.

Extractable heme was measured as explained in Materials and Methods. Values

are mean ± SD (n ¼ 3). An asterisk denotes significant difference from control

(p < 0.05).

144 RAEL ET AL.



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stained with Coomasie blue, indicating that no significantprotein degradation or cross-linking had occurred (data notshown). Probing a Western blot of erythrocyte membraneproteins with anti-human Hb produced no signal, indicatingthat no Hb adducts had been formed with membrane proteins(data not shown).

DISCUSSION

Arsine (AsH3) has been recognized as a potent hemolyticagent for well over 100 years (reviewed by Fowler and

Weissberg, 1974); however, the mechanism responsible for

toxicity remains unknown. Although the mechanism is not

FIG. 2. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of arsine-treated human hemoglobin. Using the incubations

from the heme-release experiment, oxyHb (A), metHb (B), and carboxyHb (C) were analyzed by MALDI-TOF MS. Controls are the top spectra, and AsH3-treated

samples are the bottom spectra. The theoretical molecular weight of native heme is 616.5 Da.




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known, it is clear that oxyHb is required for induction ofhemolysis (Rael et al., 2000). The purpose of this study was toinvestigate the interaction of AsH3 with Hb in order todetermine if exposure to AsH3 caused release of free heme,produced As–Hb complexes, or induced cross-links of Hb witherythrocyte membrane proteins.

Hatlelid et al. (1996) reported formation of metHb, hemi-chromes, and Heinz bodies (protein precipitation, verifiedspectrophotometrically) when AsH3 was incubated with iso-lated oxyHb. Blair et al. (1990) also showed the production ofmetHb and Heinz bodies in circulating erythrocytes of ratstreated with AsH3. Hemoglobin oxidation has long beenrecognized as a disrupter of erythrocyte function. Chiu andLubin (1989) described the process of Hb oxidation in a seriesof steps beginning with the formation of metHb followed byhemichrome formation and breakdown to precipitated globin(Heinz bodies). During degradation of the Hb complex, thetoxic heme moiety is released, resulting in widespread damageto the erythrocyte membrane. The current study investigatedthis hemolytic scenario in AsH3-induced hemolysis. Becauseheme is the proposed toxic species released during hemoglobindegradation, free heme was measured directly using a protocolbased on the assay described by Letarte et al. (1993).

Treatment of human oxyHb with AsH3 increased the releaseof free heme by nearly 1 lM from a 0.4 mM oxyHb solution.Because the normal concentration of hemoglobin in intacterythrocytes is about 15 times higher than that used in ourstudy, it would be reasonable to assume that the concentrationof free heme released in erythrocytes treated with AsH3 wouldbe significantly greater than 1 lM. Incubation of normal mouseerythrocytes with as little as 1 lM ferriprotoporphyrin IX(hemin) has been shown to cause potassium loss and swellingwithin minutes, and concentrations above 2 lM cause hemo-lysis (Chou and Fitch, 1981). Previous studies have demon-strated that formation of carboxyHb in erythrocytes preventsAsH3-induced hemolysis (Rael et al., 2000). Therefore, if hemerelease is involved in the hemolytic mechanism of AsH3, treat-ment of carboxyHb with AsH3 should not affect heme release.Indeed, treatment of human carboxyHb with AsH3 had no effecton heme release. Taken together, these results suggest thatrelease of free heme may be responsible for the colloid-osmotictype hemolysis produced by AsH3. However, colloid-osmotictype hemolysis is also produced by sulfhydryl reagents, such asN-ethylmaleimide (NEM) (Jacob and Jandl, 1962).

The present data suggest that AsH3 somehow weakens theinteraction between the globin chains and the heme molecule,leading to heme release. There are several possible explan-ations for the AsH3-induced increase in heme release and itseffect on the overall hemolytic mechanism of AsH3 on eryth-rocytes. One possibility is that arsenic, binding to the sixthligand position of the Fe2þ of heme, decreases the strength ofthe bond between heme and histidine on the globin. Binding ofligands, such as CO and CN�, increase the stability of the hemeby increasing the strength of this bond (Traylor and Sharma,

1992; Hargrove et al., 1994). If arsenic forms a bond with Fe2þ,it may weaken the bond with the histidine on globin andincrease heme release. This idea is supported by the observa-tion that treatment of carboxyHb with AsH3 had no effect onheme release, indicating that the nature of the sixth Fe2þ ligandis important in the effect on heme release. Matrix-assisted laserdesorption/ionization-MS analysis of heme from AsH3-treatedHb, however, did not provide evidence for arsenic binding toheme iron. However, the increased frequency of methyl acetateion fragment (�CH2OOH, m/z ¼ 59) loss from oxyHb treatedwith AsH3 indicates that the reaction with AsH3 may havealtered electron distribution in the heme group such that thebond strengths at these sites are lower.

A second possible explanation is that AsH3 modifies theglobin chain or heme moiety in a way that decreases thestrength of heme binding. Because AsH3 is a highly reducedform of arsenic that appears to be oxidized during the reactionwith hemoglobin (Carter et al., 2003), it could cause a confor-mational change in the Hb molecule by reducing the globinchains or the porphyrin rings, which results in decreasedaffinity of the globin for heme. Another possibility is thatAsH3, or a breakdown product of AsH3, binds directly to theglobin chains, resulting in modified protein conformation andeventual precipitation. Multiple targets exist on the Hbmolecule that are susceptible to arsenic adduction; however,cysteine residues are the most likely targets. In human Hb, thea-chain contains one cysteine residue, whereas while the b-chain has two cysteine residues within its amino acid sequence(Lehninger et al., 1993). Therefore, there are six sulfhydrylgroups per Hb molecule, of which two are readily reactive withmetallic sulfhydryl inhibitors (Ingram, 1955). Previous studieshave shown that lewisite (dichloro(2-chlorovinyl) arsine) andphenyldichloroarsine form adducts with cysteine on the b-globin chains of human Hb (Fidder et al., 2000; Chong et al.,1989). These sulfur groups are clearly important in hemeretention, as evidenced by increased heme release fromglobins treated with NEM (Scheler et al., 1963). Modificationof the two b-globin thiols also increased the rate of auto-oxidation and precipitation in isolated Hb solutions (Allenand Jandl, 1961). In the present study, no modifications of thea- or b-globin chains were found by mass spectrometry,suggesting that exposure to AsH3 does not cause significantchanges in the Hb molecule. However, AsH3-induced adductsmight be labile and susceptible to neutral loss within the massspectrometer.

This finding has implications for the proposed formation ofAs-Hb adducts following exposure to AsH3. It is clear thatsome arsenic is tightly associated with erythrocytic proteinsafter AsH3 exposure, being present in a complex that is strongenough to resist decomposition during dialysis (Graham et al.,1946). The lack of arsenic binding to purified Hb in the presentstudy suggests that other cellular components are required toconvert AsH3 to the form that ultimately binds to Hb, or that Hbitself is not the site of arsenic binding in these experiments.

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Finally, the hypothesis concerning the generation of hemo-globin–skeletal protein complexes as part of the mechanismof AsH3-induced hemolysis was also investigated. Accordingto Fortier et al. (1988), this complex is formed after theoxidation of Hb, which increases the affinity of Hb forsulfhydryl-containing, cytoskeletal components. It was con-cluded that a quantitative relationship exists between themembrane-associated protein complex and increased mem-brane rigidity. It has been demonstrated that increasing in-tracellular calcium concentration increases the amount ofmembrane-attached Hb (Friederichs et al., 1992). Based onthe observations that AsH3 interacts with sulfhydryl groups andincreases intracellular calcium concentrations in erythrocytes(Rael et al., 2000), the possibility of the formation of sucha complex in the case of AsH3 was explored. One of theobvious cytoskeletal targets is spectrin, which has been shownto have globin-binding domains (Bhown et al., 1989). Also, thesulfhydryl inhibitor NEM has been shown to selectively altererythrocyte deformability via cross-linking of sulfhydrylgroups located on spectrin (Fischer et al., 1978). A compoundknown to produce hemoglobin–cytoskeleton complexes ishydrogen peroxide. Snyder and co-workers (1988) showedthe formation of such a complex verified by Western blot inerythrocytes treated with hydrogen peroxide. Pretreatment oferythrocytes with NEM resulted in decreased lipid peroxida-tion and spectrin–hemoglobin cross-linking, as well as lessmarked alterations in cell shape and membrane deformability.Equally important was the finding that hydrogen peroxidecaused no significant decrease in intracellular glutathione(GSH). In these experiments, no such complex was observed,even when the cells were treated with hydrogen peroxide. Thisresult may be a false negative because hydrogen peroxide, usedas a positive control, also failed to induce hemoglobin–proteinadducts. The reason may be due to the antibody, whichrecognizes intact Hb tetramers and possibly globin chains.Hemoglobin in its native form does not bind to spectrin, butwhen denatured, it exhibits a strong binding (Bhown et al.,1989). Based on the Western blot findings presented herein, Hband its globin components do not appear to form complexeswith the erythrocyte membrane in the presence of AsH3.Because of the increase in heme release in isolated Hb treatedwith AsH3 demonstrated in this study, the possibility of heme–membrane complexes in erythrocytes should be probed.

In conclusion, AsH3 caused a significant increase in hemerelease from isolated oxyHb, but it had no effect on carboxyHband metHb. There was no in vitro formation of arsenic-Hbadducts or globin chain modifications as evidenced by electro-spray mass spectrometry although the heme MALDI-MSfragmentation pattern changed when AsH3 was added. Usingan antibody that detects Hb only, no Hb–cytoskeletal proteinadducts were observed in erythrocytes treated with AsH3. Thisdoes not rule out the possibility of heme interacting with theerythrocyte cytoskeleton. Based on the data presented in thisstudy, further evaluations of the interactions of heme with the

erythrocyte membrane could potentially determine the mech-anism of hemolysis caused by AsH3.

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