BIOCHEMICAL MEDICINE AND METABOLIC BIOLOGY 36, 60-69 (1986)

The Cytotoxic Activity of Hematoporphyrin: Studies on the Possible Role of Transition Metals

HUA LIN AND JOHANNES EVERSE

Department of Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430

Received March 4, 1985

Hematoporphyrin, when activated by light, becomes toxic to many cells. Fur- thermore, when injected into tumor-bearing animals, hematoporphyrin is pref- erentially retained by the tumor cells. Because of these properties hematoporphyrin has been used as an anticancer agent (l-3).

The mechanism of the photoactivation of hematoporphyrin has been extensively studied (for a review see Ref. (4)). Considerable evidence suggests that the toxic activity of photoactivated hematoporphyrin is mediated by some form of activated oxygen. Both singlet oxygen and hydroxyl radical have been implicated as possible intermediates of the reaction.

A number of other compounds are capable of causing damage to cells or cellular components, reportedly by means of active oxygen species. These compounds include hemin (5), various peroxidases (6-E), and various metal ligands (13- 18). Unlike hematoporphyrin, each of these compounds contains a liganded metal ion. Although activation of appropriate receptors by light is known to lead to electronically excited molecules, which could transfer their energy to molecular oxygen to form singlet oxygen, single electron oxidations or reductions (i.e., the formation of free radicals) almost exclusively involve the participation of a metal ion. In accordance with this, the central role of the liganded metal ions in the toxic activity of the compounds mentioned above has been amply demonstrated (5-18).

We have previously observed that hemin in the.presence of ascorbate promotes the lysis of erythrocytes in an oxygen-dependent reaction.’ It seemed logical to expect that hematoheme under similar conditions would also possess cytotoxic properties toward erythrocytes and this was indeed found to be the case (see Results). A question then arises as to a possible participation of a metal ion in the light-activated hematoporphyrin reaction. Several observations further suggest such a mechanism. First, Whitten (19) has shown that illumination of hemato- porphyrin in the presence of various metal ions and in the absence of oxygen

’ J. C. Brady, S. J. McFaul, and J. Everse, unpublished results.

60 0885-4505186 $3.00 Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

CYTOTOXIC ACTIVITY OF HEMATOPORPHYRIN 61

can lead to the incorporation of the metal ions and the formation of hematohemes. The possibility thus exists that the illumination of hematoporphyrin in the presence of cells may lead to the incorporation of spurious iron atoms into the porphyrin, especially when the hematoporphyrin is bound to the lipid membranes where oxygen tension is very low.

Second, Buettnes and Oberley (20,21) have shown that light activation of hematoporphyrin leads to superoxide production. All known biological reactions in which molecular oxygen is reduced to superoxide as well as in which super-oxide is converted to hydroxyl radicals or hydrogen peroxide involve liganded transition metals.

The involvement of contaminating metal ions in the cytotoxic activity of light- activated hematoporphyrin is thus a distinct possibility and to the best of our knowledge a metal-mediated mechanism has never been conclusively ruled out. We therefore decided to address this question. The experiments described in this article were designed to obtain evidence for or against the participation of Fe or Cu in the cytolytic activity of light-activated hematoporphyrin toward erythrocytes.

MATERIALS AND METHODS

Materials. Hematoporphyrin, nicotinamide, uric acid, ascorbic acid, 2,4- dinitrophenol, EDTA, and diethylenetriaminepentaacetic acid were purchased from Sigma Chemical Company (St. Louis, MO.). All other reagents were purchased in reagent grade quality from national suppliers.

Erythrocytes were prepared from freshly drawn rabbit blood. One milliliter of Na-heparin solution (1000 u/ml; Lypho-Medical, Inc.) was added to each 4 ml of blood. The blood was then diluted lo-fold with phosphate-buffered saline (9.5 mM Na-phosphate, 140 mM NaCl) (PBS), pH 7.4, and centrifuged at 3000 rpm for 5 min. The supernatant was discarded and the erythrocytes were washed twice more with PBS. The final erythrocyte pellet was suspended in PBS and the number of erythrocytes/ml was determined by counting an appropriate dilution in a hemocytometer.

Assay for cytolytic activity. The cytolytic activity of hematoporphyrin was determined using the continuous turbidometric method developed in our laboratory (22). The sample cuvettes contained each of the components of the cytolytic system; the control cuvette contained all ingredients, except hematoporphyrin. The amounts used of each component are indicated in the figure legends and the total assay volume was 1 ml. Both cuvettes were placed in a light box (Bio- Rad Laboratories, Model 300 LS) for the indicated length of time and immediately transferred to a Beckman Model 24 spectrophotometer. The position of the cuvettes was reversed, in that the- sample cuvette was placed in the reference cuvette holder and the reference cuvette was placed in the sample cuvette holder. Because of this reversed arrangement, the decrease in turbidity, monitored at 600 nm, was registered as an increase in absorption. This was necessary in order to avoid reading negative absorbances (22).

The cytolytic activity of hematoheme was determined using the same method by adding hematoheme (final concentration: 0.1 mM), ascorbic acid (final con-

62 LIN AND EVERSE

centration: 1 mM), other compounds as indicated, and 1 x lo6 erythrocytes to phosphate-buffered saline, pH 7.4, to a final volume of 1.0 ml. The arrangement of the cuvettes in the spectrophotometer was the same as described above.

Rates of cell lysis were determined from the slope of the curve during the time that active cell lysis was taking place.

Preparation of hematoheme. Hematoheme was prepared from hematoporphyrin with the method described by Inubushi and Yonetani (23) for the incorporation of Fe into porphyrins, except that the final purification step (A1203 absorption) was omitted. The hematoheme preparation was judged to be free of hematoporphyrin by two criteria: the lack of any hematoporphyrin fluorescence and the lack of any cytotoxic activity following illumination.

RESULTS

Lysis of Erythrocytes by Hematoporphyrin

Figure 1 illustrates the changes in absorption at 600 nm observed when rabbit erythrocytes are lysed by hematoporphyrin. The reaction proceeds with an initial lag time of several minutes during which no cell lysis occurs, followed by a period where cells lyse at a measurable rate until maximum cell lysis has occurred. The lag time as well as the rate of cell lysis is dependent on the concentration of hematoporphyrin, the concentration of erythrocytes, and the duration of exposure to light (data not shown). Similar toxicity profiles of hematoporphyrin toward mammalian cells have previously been described (4,24,25).

Figure 2 illustrates the rate of cell lysis as a function of hematoporphyrin concentration. These data indicate that the dependence of the rate on hemato- porphyrin concentration displays saturation kinetics, suggesting that a secondary reaction becomes rate-limiting at higher hematoporphyrin concentrations.

No lysis occurred under any conditions when the buffer was first deoxygenated with N2 prior to use. If, however, air was bubbled through the deoxygenated

FIG. 1. Spcctrophotometric determination of erythrocyte lysis by hematoporphyrin. Assay contained 10 ELM hematoporphyrin and 2 x lo6 rabbit erythrocytes in 1 ml phosphate-buffered saline, pH 7.4. Light exposure= 45 sec. Wavelength= 600 nm.

CYTOTOXIC ACTIVITY OF HEMATOPORPHYRIN 63

002 0.04 006 0.08

[ Hemotoporphyrin I. mM

FIG. 2. Rate of erythrocyte lysis as a function of hematoporphyrin concentration. Cuvettes contained the indicated amount of hematoporphyrin and 1 x lo6 erythrocytes in 1.0 ml phosphate-buffered saline, pH 7.4. The assays were illuminated for 45 sec. Rates were calculated from the linear portion of the obtained lysis profile.

reaction mixture, and the mixture was reexposed to light, the normal lytic reaction was observed. These observations confirmed that the presence of oxygen is a requirement for the cytotoxic reaction of hematoporphyrin (4, and references therein).

The cytolytic activity of hematoporphyrin is also known to have an absolute requirement for light. Under our assay conditions the cytolytic rate was found to be linearly propo~ional to the time of exposure for at least up to 60 set (Fig. 3).

These results, which mostly confirm findings made by other investigators using different techniques, were obtained to demonstrate that the continuous assay can appropriately be used to determine the cytolytic activity of the porphyrins.

0 IO 20 30 40 50 60 Time of Light Exposure, set

FIG. 3. Relationship between rate of erythrocyte lysis and exposure to light. Conditions were the same as in Fig. 1, except that 20 PM hematoporphyrin was used.

64 LIN AND EVERSE

When erythrocytes were added immediately after the illumination of the he- matoporphyrin solution, no cell lysis was observed. Similarly, illumination of the hematoporphyrin and the erythrocytes simultaneously but in separate vessels, followed by combining the solutions, did not promote cytolysis. These results confirm that the toxic intermediate formed by the illumination of hematoporphyrin has a very short half-life, since a few seconds after illumination the hematoporphyrin no longer possessed cytolytic activity (the half-life of singlet oxygen and hydroxyl radicals is a few microseconds).

Effect of Pyridine

Pyridine is known to facilitate the incorporation of metal ions into porphyrins (4,26). We therefore investigated the effect of pyridine on the cytolytic activity of hematoporphyrin. As shown in Fig. 4, pyridine accelerated the rate of cell lysis, reaching a maximum of about threefold activation at a pyridine concentration of 1% v/v (0.125 M). These results could be explained in support of the concept that illumination of hematoporphyrin catalyzes the incorporation of a metal ion into the hematoporphyrin ring.

Addition of Metal Ions

To further investigate the possibility that the observed cytotoxic activity was caused all or in part by spurious metal ions, the effect of added metal ions on the rate of cell lysis was tested. Neither iron (FeCl,) nor copper (CuC&), when added to the reaction mixture prior to light exposure, at concentrations up to 0.1 mM had any effect on the rate of cell lysis. Furthermore, EDTA and di- ethylenetriaminepentaacetic acid at 1 mM did not affect the profile of the cytolytic reaction. These results suggest that spurious metal ions did not play a significant role in the cytolytic reaction.

Incorporation of Metal Ions by Light

Hematoporphyrin is a fluorescent compound, whereas hematoheme is not. The incorporation of metal ions into hematoporphyrin can therefore conveniently be measured by following the decrease in hematoporphyrin fluorescence. Hema-

[Pyrldine 1, %

FIG. 4. Effect of pyridine on the rate of erythrocyte lysis. Conditions were the same as in Fig. 1, except that pyridine (v/v) was added to the cuvette immediately prior to illumination.

CYTOTOXIC ACTIVITY OF HEMATOPORPHYRIN 65

toporphyrin (10 PM) was mixed with 10 PM FeCl, in 9.5 mM phosphate buffer, pH 7.4, and illuminated for up to 10 min, at temperatures between 25 and 44°C. Mixtures were illuminated under atmospheric conditions as well as under NZ, in the presence and absence of erythrocyte membranes and in the presence and absence of 1% pyridine. In none of these experiments could we detect any decrease in the hematoporphyrin fluorescence. We concluded that no detectable amounts of hematoheme are formed under these conditions.

Effect of Various Compounds on the Cytotoxic Activity of Hematoporphyrin and Hematoheme

If the cytotoxic activity of activated hematoporphyrin results from the incor- poration of a metal ion and therefore, in fact, represents the cytotoxic activity of hematoheme, one would expect that compounds affecting the cytotoxicity of hematoporphyrin would have a similar effect on the cytotoxicity of hematoheme. To investigate this point we tested the effects of uric acid, ascorbate, 2,4-dini- trophenol, and pyridine on both cytolytic reactions (Table 1).

The hematoporphyrin was potently inhibited in the presence of uric acid, yielding a Ki of about 300 PM (Fig. 5). However, 300 PM uric acid had no effect on the hematoheme reaction. Ascorbate also inhibited the cytolytic activity of hematoporphyrin; however, this compound serves as a substrate in the hematoheme reaction. Pyridine activated the cytolytic activity of hematoporphyrin as described above, but strongly inhibited the hematoheme-mediated cytolysis (0.2% pyridine inhibited the reaction 70%). Dinitrophenol at 0.1 mu slightly inhibited the cytolytic activity of hematoheme (20%), whereas it potently inhibited the hematoporphyrin reaction (Ki = 30 PM).

The data presented in Table 1 clearly demonstrate a different and, in some cases, an opposite response of the two cytolytic reactions to the four agents described. These results therefore strongly suggest that the cytolytic reaction promoted by hematoporphyrin proceeds with a mechanism different from that promoted by the hematoheme.

TABLE 1 Effect of Various Compounds on the Cytolytic Activities of Hematoporphyrin and Hematoheme

Compound Hematoporphyrin Hematoheme

Uric acid

Ascorbic acid

Pyridine

Dinitrophenol

Inhibition (K, = 300 jLM)

Inhibition (K, = 200 /.LM)

Activation

Strong inhibition (K, = 30 /AM)

No effect

Substrate

Inhibition (K; = 17 rnM)

Weak inhibition (K, > 1 rnM)

Note. Assay mixtures contained 10 PM hematoporphyrin, 2 x lo6 rabbit erythrocytes, and varying concentrations of the inhibitors in 1 ml phosphate-buffered saline, pH 7.4. Light exposure: 45 sec.

66 LIN AND EVERSE

q , , .

0 0.1 0.2 0.3 I

[ Urote 1. mM

FIG. 5. Inhibition of erythrocyte lysis by uric acid. Conditions were the same as in Fig. 1, except that uric acid was added immediately prior to illumination.

DISCUSSION

Various lines of evidence have been presented indicating that the toxic activity of photoactivated hematoporphyrin is mediated by singlet oxygen (4). However, the excitation of hematoporphyrin has also been shown to lead to superoxide production (20,21). Superoxide in turn can give rise to hydroxyl radicals (27,28), which have also been implicated as the species responsible for the hematoporphyrin toxicity (4). However, all known biological reactions in which molecular oxygen is reduced to superoxide as well as in which superoxide is converted to hydroxyl radicals involve liganded transition metals. Similarly, the production of singlet oxygen in the peroxidase reaction (10,29) involves the participation of a liganded metal ion. In considering the oxygen dependence of the photoactivated hema- toporphyrin reaction, the ability of molecular oxygen to bind to hemes and heme proteins by coordinating to the transition metal ion is of course well known.

Thus, the mechanism by which molecular oxygen is converted to singlet oxygen and/or hydroxyl radicals by an electronically excited hematoporphyrin ring is not immediately apparent. On the other hand, the generation of such active oxygen species can be readily explained if a transition metal ion were incorporated into the hematoporphyrin ring during photoactivation. Whitten (19) showed that such an incorporation can indeed take place during photoactivation of hematoporphyrin.

Our present observations, however, invalidate such a mechanism by four criteria. First, no evidence for the incorporation of metal ions could be obtained. No decrease in fluorescence was observed when hematoporphyrin was illuminated in the presence of Fe and Cu ions, the metal chelators EDTA and diethylene- triaminepentaacetic acid did not affect the reaction, and no activity could be detected when a hematoporphyrin solution was tested for cytolytic activity in the presence of ascorbate several minutes after the solution was illuminated. Second, the addition of metal ions did not increase the cytolytic activity as would be expected if the reaction were dependent on contaminating metal ions. Third,

CYTOTOXIC ACTIVITY OF HEMATOPORPHYRIN 67

the hypothesis could only be valid if hematoheme is a substantially better cytolytic agent than hematoporphyrin; our results indicate, however, that hematoporphyrin is a better cytolytic agent than hematoheme on an equimolar basis. Fourth, the mechanism of the cytolytic reaction of hematoporphyrin appears to be different from that catalyzed by hematoheme as demonstrated by the effect of various inhibitors and activators.

Although the results presented in Table 1 clearly rule out the possibility that hematoheme is the active component in the photoactivated hematoporphyrin reaction, they raise some interesting questions as to the mechanism of the he- matoporphyrin reaction.

Uric acid has been described as an excellent scavenger of singlet oxygen (30,31). The inhibitory effect of uric acid on the cytolytic activity of hematoporphyrin therefore supports the concept that this activity is mediated by singlet oxygen. However, the lack of any effect of uric acid on the hematoheme activity must then be interpreted to indicate that singlet oxygen is not an intermediate in this reaction. It should be noted, however, that uric acid is not specific for singlet oxygen; therefore these results should not be interpreted as a means of identifying the active intermediate. Pyridine readily ligands to the fifth and sixth coordinates of Fe-containing porphyrins (32) and therefore would be expected to inhibit any reaction involving the metal ion. Accordingly, pyridine was found to inhibit the cytolytic activity of hematoheme. However, pyridine activates the hematoporphyrin reaction. The reasons for this activation are not immediately apparent; but the fact that the activation shows saturation kinetics could mean that pyridine is stabilizing an unstable intermediate.

Dinitrophenol is a compound that can readily react with free hydroxyl radicals or superoxide to form a semiquinoid radical. Its strong inhibitory effect on the hematoporphyrin reaction could be construed to indicate that superoxide is an intermediate in the cytotoxic action and could be the source of the singlet oxygen and/or hydroxyl radicals. Again, the lack of specificity of dinitrophenol prevents one from making any definite conclusions as to the nature of the intermediate.

Nevertheless, the results presented in Table 1 indicate that the effects of several unrelated compounds on the cytotoxic activity of photoactivated hematoporphyrin and of hematoheme vary widely, and in some cases opposite effects are observed. These data can only be interpreted as indicating that the two cytolytic reactions proceed by two different mechanisms, involving different intermediates. The fact that both reactions lead to a time-dependent destruction of the erythrocyte mem- brane must then indicate that more than one intermediate can initiate such damage.

To date, the formation of one or more active oxygen species has been proposed in an increasing number of reactions, involving compounds of widely different chemical structures, as indicated above (5-18). Since the number of different active oxygen species that can be generated is obviously limited, one would anticipate that the same active species may be generated by several different compounds. Comparative studies using appropriate inhibitors as used here, may be useful to further substantiate this point.

68 LIN AND EVERSE

SUMMARY

Hematoporphyrin acquires a potent cytolytic activity toward erythrocytes when activated by visible light. Considerable evidence has been obtained suggesting that this toxic activity is mediated by certain active oxygen species, including singlet oxygen and hydroxyl radicals. These active oxygen species have also been proposed as intermediates in the toxic activity of peroxidases, hemin, and a variety of metal complexes. Unlike hematoporphyrin, all these compounds contain a liganded Fe atom, which appears to play a central role in the activation of molecular oxygen. In order to ascertain whether the generation of active oxygen by hematoporphyrin may also involve the participation of a metal ion we have compared the cytolytic activity of hematoporphyrin with that of he- matoheme. The participation of a metal ion in the light-activated hematoporphyrin reaction was ruled out on the basis of four criteria: (1) no increase in cytolytic activity was observed upon the addition of Fe or Cu ions; (2) no evidence could be obtained for the incorporation of a metal ion into hematoporphyrin during light activation; (3) hematoporphyrin is a more potent cytolytic agent than he- matoheme on an equimolar basis; and (4) the activities of the two cytolytic agents are affected differently by various activators and inhibitors of the toxic reaction. Our results further indicate that the mechanism of the cytolytic activity promoted by light-activated hematoporphyrin is distinctly different from that promoted by hematoheme in the presence of ascorbate. We conclude that the two cytolytic reactions are most likely propagated by two different forms of active oxygen.

ACKNOWLEDGMENT

This work was supported by Grant CA32715 of the National Cancer Institute, U.S. Public Health Service.

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