The Degradative Effects of Porphyrins and Heme Compounds on Components of the Microsomal Mixed Function Oxidase System%

The effects of in vitro treatment of the hepatic microsomal fraction with various porphyrin compounds on the activity and the content of the heme-containing components of the mixed function oxidase system were studied. The compounds examined were hematin, methemalbumin (with heme to protein molar ratio of 13:l or l:l), mesohemalbumin, bilirubin, biliverdin, mesoporphyrin IX, and protoporphyrin IX. The activity of the system was monitored by measuring its oxidative activity for the type I and type II substrates, ethylmorphine and aniline, respectively; as well as the microsomal contents of cytochrome P-450 and b, and %-labeled heme. Mesoporphyrin IX was found to be most effective in inhibiting the oxidative activity of the mixed function oxidase system as well as in decreasing the microsomal contents of cytochromes P-450, b,, and heme. Biliverdin exerted no effect on these parameters. Hematin and the other compounds studied exerted variable inhibitory effects on the system. The degradative and inhibitory effects of protoporphyrin IX and mesoporphyrin IX could be blocked significantly by conducting the studies in the dark. The presence of biliverdin decreased the inhibitory effects of the porphyrins on the system; conversely the effects could be magnified in

The effects of in vitro treatment of the hepatic microsomal fraction with various porphyrin compounds on the activity and the content of the heme-containing components of the mixed function oxidase system were studied. The compounds examined were hematin, methemalbumin (with heme to protein molar ratio of 13:l or l:l), mesohemalbumin, bilirubin, biliverdin, mesoporphyrin IX, and protoporphyrin IX. The activity of the system was monitored by measuring its oxidative activity for the type I and type II substrates, ethylmorphine and aniline, respectively; as well as the microsomal contents of cytochrome P-450 and b, and %-labeled heme. Mesoporphyrin IX was found to be most effective in inhibiting the oxidative activity of the mixed function oxidase system as well as in decreasing the microsomal contents of cytochromes P-450, b,, and heme. Biliverdin exerted no effect on these parameters. Hematin and the other compounds studied exerted variable inhibitory effects on the system. The degradative and inhibitory effects of protoporphyrin IX and mesoporphyrin IX could be blocked significantly by conducting the studies in the dark. The presence of biliverdin decreased the inhibitory effects of the porphyrins on the system; conversely the effects could be magnified in the presence of deuterium oxide. It is suggested that the mechanism by which porphyrins inhibit the mixed function oxidase system is through porphyrin-sensitized photo-oxidation of various constituents of the hepatic microsomal fraction and that the formation of singlet oxygen molecules is most likely involved in this process. Moreover the destructive effects of heme compounds on the microsomal components and activities of the drug-metabolizing mixed function oxidase system raise questions concerning the hypothesis that the components of this system, and specifically cytochrome P-450, are involved in the activity of the heme oxygenase system.
The term "photodynamic" action refers to a phenomenon which encompasses the damaging effects on biological membranes or chemical structures of sensitizing compounds, oxygen, and light (l-3). Numerous examples of photodynamic actions are known; among these are included the severe tissue destruction characterizing certain types of acquired and genetic porphyrias of man which are associated with severe photosensitivity (4-7). In these disorders, it is generally accepted that light absorption by circulating porphyrins is the cause of the photosensitivity (8,9) and the resultant tissue damages seen in affected individuals.
Although the reactions of the excited porphyrins are not as yet well understood, it is known that free porphyrins and metalloporphyrins are singlet oxygen sensitizers (1, 10). Porphyrins and metalloporphyrins differ greatly in their effectiveness as sensitizers of singlet oxygen formation however, free porphyrins being in general more effective than the metalloporphyrins (10). Porphyrins and metalloporphyrins are damaging to biological systems probably due to their solubility properties; since these com-*This research was supported by United States Public Health Service Grants ES-00621 and ES-01055 and by an institutional grant from The Rockefeller Foundation for studies in reproductive physiol-WY.
pounds have a relatively low water solubility, they exist closely associated with biological membranes and form complexes with various components of the membranous proteins (11). In this complexed form the porphyrins and the metalloporphyrins are photoexcitable and are capable of inflicting photodynamic damage to the membrane structures. Since the components of the hepatic microsomal mixed function oxidase system which oxidizes drugs (12)

Assay of Mined Function
Oxidase Activity-At the end of the incubation period, the mixtures were centrifuged at 163,000 x g for 40 min. The pellets were resuspended in buffer to a protein concentration of 4 to 5 mg per ml. This suspension was used as the enzyme source for the assay of microsomal mixed function activity as well as for the spectral studies and heme determinations. "Washing" of the microsomal pellets after treatment was omitted since it was found that this process did not effectively eliminate the bound compounds. Enzyme activity for type I substrates was measured using ethylmorphine. Formaldehyde formed by demethylation of ethyl morphine was determined by the method of Nash (18). Using the incubation mixture described earlier (19), the enzyme activity for type II substrates was measured using aniline. Aniline hydroxylase activity was determined as described by Imai et al. (20). In the studies in which the influence of after centrifugation (163,000 x g, 40 min) the microsomal pellets were resuspended in buffer (4 to 5 mg of protein/ml), and heme was extracted by treatment of the microsomal suspension with ethyl acetate-acetic acid (4:l v/v) as described by Schwartz et al. (22) for 1 hour at room temperature.
The ethyl acetate layer was washed repeatedly with 7% NaCl and once each with 3 N HCl and 7.5 N HCl. Microsomal heme was recovered from the organic phase by the addition of petroleum ether (b.p. 30-60") in excess followed by centrifugation.
The hemin crystals thus obtained were taken into chloroform, aliquots were removed, concentrated under air, and counted as above.
Spectral Studies-Cytochrome P-450 content was determined from the reduced-minus CO-difference spectrum using NADPH as the reducing agent and an extinction coefficient of 91 mM-km-' between 450 and 490 nm (23). Cytochrome b, content was measured using NADH as the reducing agent and an extinction coefficient of 185 mM-'cm-'between 412 to 425 nm (24). The concentration of heme was determined by the pyridine-hemochromogen method as described by Paul et al. (25) using the sodium dithionite-reduced minus oxidized difference spectrum between 557 and 575 nm and an extinction coefficient of 32.4 rnM-'cm-'. All spectral studies were conducted using an Aminco Chance DW-2 spectrophotometer. Preparation  Table  I shows the effect of treatment of microsomal fractions (1.5 to 2.5 mg of protein/ml, at 37" for 30 min, under atmosphere, in light) with methemalbumin (having a heme to protein molar ratio of 13:l or 1:l) and mesohemalbumin, at final concentrations of 17 and 34 pM. As shown, in all treatments there was a significant decrease in the oxidation of the type I and type II substrates, ethylmorphine and aniline. Generally all treatments had a greater effect on the oxidation of the type I substrate than on the type II substrate. Similarly there was a greater decrease in the cytochrome P-450 content of the microsomal fraction than in the content of cytochrome b,. The nonspecific binding of heme to microsomal proteins was observed when the microsomal fraction was pretreated with methemalbumin with a heme to protein molar ratio of 13: 1, as reflected in the increase in total microsomal heme content. In contrast when the added heme was in a totally complexed form, as in methemalbumin with a 1:l molar ratio, no increase in microsomal heme content was noted. Table II shows that when the hepatic microsomal fractions (1.8 to 2.5 mg of protein/ml) were preincubated with various concentrations of hematin (37", 30 min, air, light) ranging between 8.5 to 34 PM there was a decrease in the oxidative activity of the microsomal fraction. Again enzymatic activity for the type I substrate was affected more significantly than that for the type II substrate. However, there did not appear to be a direct relationship between the concentration of hematin utilized and its effect on enzyme activity or on the hemoprotein content of the microsomal fraction. At the end of incubation time the microsomal fractions were centrifuged (163,000 x g, 40 min) and resuspended in buffer (4 to 5 mg of protein/ml).
Microsomal enzyme activities for the type I and type II substrates, ethylmorphine and aniline, respectively, were assayed as described in Table I. The microsomal content of cytochrome P-450 was determined from the NADPH-reduced minus CO-difference spectrum, and the content of cytochrome b, was measured using NADH as the reducing agent. Thereafter the incubation mixtures were centrifuged, microsomal fractions were resuspended, and assays for enzymatic activity as well as spectral studies were conducted. As Table III indicates, the preincubation of microsomal fractions with biliverdin had no effect on any of the measured variables of the mixed function oxidase system. In contrast, the treatment of microsomal fractions with mesoporphyrin IX virtually abolished enzymatic activity as well as the heme-containing microsomal constituents and diminished the total microsomal heme content by nearly 75%. Bilirubin and protoporphyrin IX both significantly inhibited the enzymatic activity of the microsomal fraction, with bilirubin most effectively inhibiting type II activity and protoporphyrin IX being For determination of the cytochrome P-450 content, NADPH was used as the reducing agent in order to avoid the spectral interference of the reduced CO-complexed exogenous heme which was obtained when sodium dithionite was used as the reducing agent. Fig. 1 shows the absorption spectrum of the hematin-treated (17 pM) microsomal fractions. As shown, there was a great decrease in the microsomal content of cytochrome P-450 as well as an increase in the absorption at about 420 nm of the reduced CO-difference spectrum of the hematin-treated microsomes.
Effect of Porphyrins, Biliverdin, and Bilirubin on Various Parameters of Hepatic Microsomal Mixed Function Oxidase System-Rat hepatic microsomal fractions were incubated more effective against type I oxidation.
In addition both compounds decreased the content of microsomal cytochrome b,; and protoporphyrin IX diminished the cytochrome P-450 content by more than 50%. Fig. 2 illustrates the reduced CO-difference spectrum of the microsomal fraction following pretreatment with bilirubin or biliverdin. Although these compounds did not alter the microsomal content of cytochrome P-450, following bilirubin treatment no cytochrome P-420 was detectable in the microsomal preparation.  Thereafter, the "C-labeled degradation products of microsomal heme were extracted with a chloroform-methanol (2:l) mixture.
As Table  IV   Recently it has been reported that components of the mixed function oxidase system (12) which metabolizes drugs, and specifically the terminal oxidase of the system, cytochrome P-450, participate in the oxidation of various metalloporphyrin complexes (28,29). Since porphyrin compounds have been shown to be effective photosensitizers and to cause severe photodynamic damage to cellular components, it seemed of interest to investigate the effects of these compounds on different parameters of the mixed function oxidase system.
It was found that preincubation of microsomal fractions with various metalloporphyrin compounds such as hematin, methemalbumin, and mesohemalbumin, as well as porphyrins such as protoporphyrin IX or mesoporphyrin IX greatly inhibited the enzymatic activity as well as contents of specific heme-containing microsomal constituents and total microsomal heme. Mesoporphyrin IX was the most active porphyrin compound found in this study and virtually abolished microsomal enzyme activity and content of hemoproteins.
The degradative effects of the free porphyrins on all measured parameters of the mixed function oxidase system provide an alternative explanation for the observation made by others (13) that protoporphyrin IX and mesoporphyrin IX are not oxidized by the heme oxygenase system. Porphyrins, in the presence of oxygen and light, have deleterious effects on cellular constituents and their failure to be oxidized by microsomal preparations may not merely be due to their being ineffective substrates for the heme oxygenase system but may result from direct membrane damage produced in the in uitro preparations in which heme oxygenase activity was studied (13).
Bilirubin, although a singlet oxygen sensitizer, is a weak one (8, 30) and did not alter the activity of the mixed function oxidase system, nor did it change the microsomal content of cytochrome P-450 (Table III and Fig. 2). In contrast to bilirubin, biliverdin has been shown to be a singlet oxygen quencher (31) and as the data in Table III and Fig. 2 indicate, it failed to affect significantly the contents of microsomal cytochromes P-450, P-420, or b,.
As previously noted, photodynamic actions of chemicals require the presence of light, which provides energy for the conversions of the sensitizer to the first excited singlet state  Table I. to 2.5 mg of protein/ml) in phosphate buffer made with water or deuterium oxide and were pretreated (37", 30 min, air, light) with hematin or protoporphyrin IX both at a final concentration of 34 pM. Following resedimentation of the microsomal fraction, the '"C-labeled degradation products of microsomal hemoproteins were extracted with chloroform-methanol mixture (2:l). As Table V  and biliverdin, as well as protoporphyrin IX and biliverdin, the inhibitory effects of these compounds on oxidative activity were largely nullified. In addition, their destructive effects on microsomal hemoproteins were greatly inhibited. In unreported studies, biliverdin also provided a significant protective effect against the deleterious effects of mesoporphyrin IX on the microsomal activity and components. However, the destructive effects of mesoporphyrin IX on microsomal enzymes were so profound that the equimolar concentration of biliverdin did not provide the full reversal of the mesoporphyrin IX effect as was observed with hematin and protoporphyrin IX. and subsequently the transition to the triplet excited state. This is the form which is involved directly in the formation of singlet oxygen or radicals which result in photodynamic damage to cellular components.
The present study indicates (Table IV) that in the absence of light the extent of photodynamic activity of porphyrin compounds, represented in terms of inhibition of enzyme activity and degradation of microsomal heme, is reduced or nullified. The observation that the inhibitory effects of hematin were not as effectively nullified as were those of protoporphyrin IX and mesoporphyrin IX suggests that the mechanism of the photodynamic action of these sensitizers may be somewhat different. This could be partly due to differences in the quantum requirement for these photosensitizers, since although the treatments with these compounds were carried out in the almost total absence of light, exposure of the preparations to some light was unavoidable during the course of the experiments.
On the basis of the finding that the lifetime of singlet oxygen is extremely sensitive to the nature of solvents (32), Merkel et al. (33) demonstrated that the lifetime of singlet oxygen in D,O was greatly prolonged over that in H,O. It has proved possible to use this property to infer the intermediacy of singlet oxygen in most photochemical reactions. The present study shows (Table V) (Table V).
Throughout this study, a general agreement was found between the decrease in enzymatic activity and the microsomal content of cytochrome P-450; but in the experiments in which this question was explored specifically, there was no stoichiometric correlation between the inhibition of drug metabolism and the amount of '"C-labeled heme degradation products produced. Similarly, in other experiments no such correlation was found between the decrease in the oxidative activity of the microsomal fraction and the amount of extractable 'C-labeled intact microsomal heme. In such experiments when the microsomal fractions containing Y-labeled heme were treated with hematin in the light, and the intact heme then extracted, a decrease of only 15% in the microsomal content of labeled heme was observed, e.g. an average (four trials) value of 25,942 dpm/ml for the control uersus 22,982 dpm/ml for the hematin-treated microsomes was obtained. These findings indicate that the destruction of microsomal heme is only one of several alterations which exogenous hematin and porphyrins produce on cytochrome P-450 and other components of the mixed function oxidase system.
The observation that hematin exerted virtually no concentration-dependent effects on the microsomal enzymes also suggests that only a limited number of exposed hematin-sensitive sites are available on the endoplasmic reticulum. Thus some constituents of the mixed function oxidase complex would be expected to come into closer contact with, or be more susceptible to, the effects of added porphyrins while others might be protected. This would be a plausible explanation of our findings, particularly in light of the fact that photodynamic actions of porphyrin compounds (because of the very short lifetime of singlet oxygen), are almost exclusively local and intramolecular rather than intersystemic. The importance of localization effects may be critical since even heme prosthetic groups have been shown to act as photosensitizers for the labile amino acids in their proximity (34,35).
The involvement of singlet oxygen formation in chemical reactions is strongly suggested by the quenching of singlet oxygen production by appropriate acceptors. Several effective singlet oxygen quenchers are known, with biliverdin being one of the few which are soluble in water (30). Accordingly, good evidence for the intermediacy of singlet oxygen in the destruction of hepatic microsomal mixed function oxidase components is provided in this study by the observations (Table VI) that in the presence of equimolar concentrations of biliverdin the effects of hematin and protoporphyrin IX on the complex were diminished significantly. Finally, it should be noted that all of the heme compounds which were found in this study to have degradative effects on microsomal components have been shown to be effective in vitro substrates for the hepatic heme oxygenase system (13). The specific involvement of cytochrome P-450 and components of the drug-metabolizing mixed function oxidase complex in the heme oxygenase system has been proposed (28). It would seem paradoxical that the substrates of an enzymatic system should be so destructive to the constituents of the system itself and in light of the findings of this study the putative role of cytochrome P-450 in the heme oxygenase system (29) should be re-evaluated.