Stomatitis

Quinacrine Enhances Vesicular Stomatitis Virus Inactivation and Diminishes Hemolysis of Dimethylmethylene Blue-phototreated Red Cells

ABSTRACT

Several photodynamic methods for virus inactivation in red blood cell (RBC) suspensions have resulted in unwanted hemolysis during extended 1-6 deg C storage. To explore the possibility that hemolysis may be mediated by a membranebound dye, a molecule similar in structure to yet different in light absorption properties from the photosensitizer was used as an inhibitor for RBC membrane binding in virus photoinactivation and photohemolysis studies. The addition of 500 (mu)M quinacrine to oxygenated RBC before treatment with 3.6 (mu)M dimethylmethylene blue (DMMB) and 219 mJ/cm^sup 2^ red light resulted in an increased extracellular concentration of the sensitizer, increased extracelluar viral inactivation kinetics, and decreased hemolysis during 1-6 deg C storage without alteration of quinacrine absorption properties. These results collectively suggest that despite its recognized affinity for viral nucleic acid, DMMB also binds to RBC membranes and that the bound dye is, in part, responsible for photoinduced hemolysis.

Abbreviations: DMMB, dimethylmethylene blue; Hb, hemoglobin; RBC, red blood cell; RPMI, Roswell Park Memorial Institute; VSV, vesicular stomatitis virus.

INTRODUCTION

Despite improvements in blood safety due to improvements in donor selection and introduction of additional infectious disease tests, a small risk of transfusion-transmitted disease remains. In the United States the risk of viral transmission per blood unit is estimated to be 1 in 1000 000 for hepatitis A virus, 1 in 138 700233000 for hepatitis B virus, 1 in 250 000-2000000 for human T-cell lymphotrophic viruses types I and II, and 1 in 10 000 for human parvovirus B 19 (1,2). With the introduction of nucleic acids testing, the risk of transmission of hepatitis C virus and human immunodeficiency virus is estimated at I in 1935 000 and 1 in 2 135 000, respectively (3).

A number of investigators have studied the feasibility of using photosensitizers in red blood cell (RBC) suspensions as a means to reduce residual infectious disease risk. Many studies have reported that one or more in vitro RBC storage properties are affected by conditions sufficient for photoinactivation of 6 logo of virus (4-11). In most cases there is a significant increase of hemolysis during extended refrigerated storage over that obtained from untreated controls. Some RBC hemolysis may be mediated by photodynamic action from a hydrophobic sensitizer bound to the RBC membrane (12).

In other studies investigators have focused on selecting dyes with high affinities for nucleic acids as a means to specifically inactivate viruses in anucleate RBC. The phenothiazine dye dimethylmethylene blue (DMMB) has been used to inactivate viruses and leukocytes in RBC suspensions (10,13,14). Based on equilibrium dialysis experiments, DMMB has been shown to bind to DNA with roughly 10 times greater affinity than the 1-2 X 10^sup -5^ M^sup -1^ binding affinity of methylene blue (15,16). The intercalation of DMMB into DNA has been demonstrated by diminution of fluorescence, redshift in the absorption maximum and induced circular dichroism when DMMB was incubated with DNA (17). Despite its affinity for nucleic acid, virucidal DMMB phototreatment of RBC suspensions results in enhanced hemolysis during 42 days of storage (10). In this study we explore the use of a compound that inhibits sensitizer binding to RBC membranes as a means to increase DMMB specificity for viruses. Quinacrine is a planar tricyclic compound with structural similarities to DMMB, yet it does not absorb red light. It binds avidly to phosphotidylethanolamine, inhibits calmodulin binding to the RBC membrane and has been used in the treatment of malaria (18-20). Here, we investigate the effects of an antimalarial drug on DMMB plus red light-induced virus photoinactivation and RBC photohemolysis.

MATERIALS AND METHODS

RBC preparation and oxygenation. Whole blood was collected in triplepack container systems (PLI-146, primary container, Baxter Healthcare, Deerfield, IL), cooled to 1-6 deg C and centrifuged at 1471 g for 4 min. After plasma expression, 110 mL of cold Erythrosol (21,22) was added to packed RBC. Blood was leukoreduced by filtration (Sepacell RZ-2000, Ashai Medical Co., Ltd., Japan, or Leukotrap(R)-SC RC. Medsep Corporation, Covina, CA).

RBC suspensions in additive solution were oxygenated to accelerate photoinactivation kinetics. Oxygenation was carried out by adding 240 mL of an O2-N^sub 2^ gas mixture (60:40%) to RBC suspensions in 1 L containers (PL2410 plastic, Fenwal, Round Lake. IL) and by subsequent incubation for 25 min at 1-6 deg C with agitation (orbital shaker, 100 rpm, 19 turn orbit, VWR Scientific, Bridgeport, NJ). Oxygen levels were measured using a blood gas analyzer (RapidLab 348, Bayer Corp., Medfield, MA) and were routinely supersaturated with levels >400 mm Hg.

Working stocks of vesicular stomatitis virus (VSV) were diluted at least 50fold into oxygenated RBC suspensions. The blood was thoroughly mixed and divided into two equal parts. To one part, a quinacrine 50 mM stock solution in Erythrosol was added, mixed and incubated at 1-6 deg C for 15 min before addition ofa 720 fLU DMMB stock solution in water. The other part was incubated at I6C for 15 min before addition of the DMMB stock solution. The final concentration of DMMB in both halves of the resulting 45% hematocrit RBC suspensions was 3.6 (mu)M. The final concentration of quinacrine in quinacrineDMMB RBC suspensions was 500 (mu)M.

In separate experiments the quinacrine stock solution was added to oxygenated RBC suspensions containing VSV. For these studies there was no subsequent addition of DMMB: the final concentration of quinacrine was 500 (mu)M of the resulting 45% hematocrit RBC suspension.

Flow-cell system. DMMB was purchased (Aldrich Chemical Company, St. Louis, MI) and purified by medium pressure liquid chromatograph as previously described (10). DMMB was incubated in RBC suspensions for 15 min at 1-6 deg C before illuminating the samples in a flow system, where blood was pumped at a rate of 0.317 mL/s by a volumetric infusion pump (Flo-Gard 6200, Baxter-Travenol, Deerfield, IL). Suspensions traveled from transfer packs through an irradiated and disposable flow cell (PL2410 plastic, Fenwal) to a receiving transfer pack ria standard blood bag tubing. The flexible flow cell was placed between two sheets of Plexiglas separated by spacers to create an illuminated 4 mL blood film of approximately 1 mm thickness. The average blood residence time within the flow cell was approximately 13 s. Two red, liquid emission diode sources (Q-beam 2001MED, Quantum Devices. Inc., Barneveld, WI) illuminated each Plexiglas sheet with 670 nm (peak intensity) +/- 13 nm (half-peak intensity) light with fluence rates adjustable up to 9.0 mW/cm^sup 2^. Fluence rates were measured using a handheld laser power meter with a silicon cell sensor at the surface of the flow cell (Edmunds Industrial Optics, Barrington, NJ).

Extracellular VSV assay. VERO cells (isolated from African green monkey kidney, CCL81, ATCC, Manassas, VA) were propagated in Roswell Park Memorial Institute medium (RPMI-1640 with glutamine, BioSource International, Rockville, MD) supplemented with 10% fetal bovine serum (Biofluids, Rockville, MD). Cells were seeded onto six-well culture plates and allowed to grow to form confluent monolayers. Control and phototreated samples were serially diluted I in 10. plated onto confluent VERO cell monolayers and incubated for 30 min with gentle rocking at 37 deg C for adsorption of the virus onto the cells. The inoculum was aspirated and washed with RPMI-1640; a semiliquid agar layer (0.2%) was added to each well. The infected monolayers were incubated at 37 +/- 2 deg C in air containing 5% CO2 for 1 day. After incubation the agar layer was removed by aspiration, and the monolayer was stained with 0.1% crystal violet in ethanol for at least 15 min. The stain was removed by aspiration, the plates washed with water and the plaques enumerated.

Hemolysis assay. Supernatant hemoglobin (Hb) was determined by the tetramethylbenzidine method (Procedure No. 527, Sigma Chemical Co., St. Louis, MO) (23). Total Hb was measured using an automated cell counter (Cell Dyn 3700, Abbott Laboratories, Abbott Park, IL).

Statistics. Determination of mean and standard deviation of experimental values and performance of two-tailed t-tests were carried out by using standard software (Instat, GraphPad Software, San Diego, CA).

RESULTS

Structure and spectral properties

The structures of DMMB and quinacrine are given in Fig. 1. Visible spectroscopy was performed on supernatants from RBC suspensions already containing either (A) DMMB and quinacrine or (B) DMMB alone, or on supernatant to which (C) DMMB and quinacrine were added or (D) DMMB alone was added. Typical spectra are given in Fig. 2. Panel I shows that treatment of RBC with quinacrine before addition of DMMB results in a supernatant (sample A) DMMB spectrum whose intensity is 90% greater than that obtained from the supernatant of RBC treated with DMMB alone (sample B). Quinacrine by itself absorbs markedly between 350 and 500 nm, and its spectra are unaffected by the presence of 3.6 (mu)M DMMB (data not shown). In panel II the addition of both quinacrine and DMMB to the supernatant (sample C) results in a 4 nm redshift, but in similar 660-664 nm peak intensities to spectra obtained from supernatant samples containing DMMB alone (sample D). Data from panels I and II collectively demonstrate that quinacrine inhibits DMMB binding to RBC. Finally, the DMMB peak intensity of supernatant derived from quinacrinecontaining RBC (panel I, spectrum A) is greater than the peak intensity when DMMB and quinacrine are added to the supernatant (panel II, spectrum C). These results suggest that the interaction of quinacrine with RBC causes DMMB to be excluded from the RBC volume and results in an increased concentration of sensitizer in the supernatant beyond that of a uniformly distributed dye.

Extracellular VSV inactivation

Results of VSV photoinactivation experiments are given in Fig. 3. RBC suspensions pretreated with quinacrine and subsequently incubated with DMM resulted in approximately 1.7-fold more rapid VSV photoinactivation kinetics than did those observed from identical units containing DMMB alone (P

Photohemolysis

RBC suspensions containing DMMB alone, DMMB and quinacrine or quinacrine alone were illuminated with 225 mJ/cm^sup 2^ red light, stored for up to 42 days at 1-6 deg C and periodically assayed for supernatant and total Hb. Results are given in Fig. 4. RBC suspensions containing DMMB and quinacrine had roughly onehalf the levels of photoinduced hemolysis observed in RBC suspensions containing DMMB alone (P

Quinacrine photostability

Spectra of diluted supernatant from red light-illuminated (225 mJ/ cm^sup 2^) and unilluminated RBC containing DMM and quinacrine are given in Fig. 5. Phototreatment of RBC under virucidal conditions did not result in any alteration in the spectrum of quinacrine, indicating that the drug does not function primarily as an antioxidant in this system.

DISCUSSION

The binding of phenothiazine dyes to RBC has been used as a surrogate marker for maintenance of RBC in vitro and in vivo properties during storage and aging, respectively (24,25). In systems for studying virus photoinactivation in RBC suspensions, cell-bound dye reduces the availability of the sensitizer for the virus and may, therefore, limit virus inactivation kinetics. Both bound and unbound dyes may contribute to photoinduced hemolysis as a result of virucidal phototreatment.

vanSteveninck and colleagues used the RBC band 3 ligand, dipyridamole, as a singlet oxygen quencher rather than as a competitive sensitizer-binding inhibitor to reduce DMMBinduced RBC membrane damage without substantial reduction of virus photoinactivation kinetics (26). We hypothesized that use of the antimalarial drug, quinacrine, with RBC might block the binding of DMMB, a phenothiazine of similar structure, to the RBC surface. Decreased cell-bound DMMB might make more dye available for interaction with the virus and thus accelerate photoinactivation kinetics. Reduced levels of cell-bound DMMB may also limit photoinduced hemolysis. Based on spectroscopic data from the RBC supernatant, quinacrine was shown to inhibit the uptake of DMMB by RBC, resulting in elevated extracellular DMMB concentrations. The increased levels of DMMB in the supernatant were greater than those anticipated if the dye were homogeneously distributed, suggesting that the membrane-bound sensitizer may serve as an intermediate for transport of the dye to the RBC cytoplasm. VSV inactivation kinetics was accelerated - 1.7-fold by the approximately two-fold increase of DMMB in the supernatant of quinacrine-treated RBC. Correspondingly, photoinduced hemolysis in quinacrine-treated cells was reduced to onehalf the levels observed in the absence of quinacrine, suggesting that RBC membrane-bound sensitizer participates in some but not all of the observed photodamage and hemolysis. Alternatively, quinacrine may protect RBC from hemolysis by its phospholipase A2 inhibitor activity, blocking the conversion of oxidized phospholipids into lysolipids (27,28).

In the absence of quinacrine, VSV photoinactivation reached a plateau at approximately 180 mJ/cm^sup 2^ (Fig. 1). The plateau effect could be caused by destruction of DMMB during the course of illumination. Perhaps quinacrine increases the concentration of extracellular DMMB so that sufficient sensitizer for virus inactivation is present throughout the irradiation period.

Although quinacrine decreases photoinduced hemolysis during storage, the levels are still greater than those desired for transfusion after 42 days of storage. In addition, the toxicological properties of quinacrine make it an unlikely candidate for use as an additive in this system (29-31). However, the general concept of using another less toxic compound to limit the extent of sensitizer binding to RBC may be useful in enhancing the specificity for virus photoinactivation in RBC suspensions.

Acknowledgements-This study was supported, in part, by grants from the National Heart Lung and Blood Institute (HL66779), Baxter Healthcare and Cerus Corporation. We appreciate the efforts of Mary StewartWesson and Chong-Son Sun, Baxter Healthcare, in supplying disposable flow-cell components and for initial characterization of the flow-cell system.

Posted on the web site on 28 August 2002.

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Stephen J. Wagner*, Andrey Skripchenko and Dedeene Thompson-Montgomery

American Red Cross Biomedical Services, Jerome H. Holland Laboratory for the Biomedical Sciences, Blood & Cell Therapy Development, Rockville, MD

Received 17 April 2002; accepted 12 August 2002

*To whom correspondence should be addressed at: Holland Laboratory, 15601 Crabbs Branch Way, Rockville, MD 20855, USA. Fax: 301-7380704; e-mail: wagners@usa.redcross.org

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