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  • XIV. Development of Photobiotechnologies

  • A. Photodynamic Herbicides

  • 1. Concept and Phenomenology

  • 2. Photodynamic Effects of Metabolic Tetrapyrroles on Isolated Chloroplasts
  • a. Fluorescence Properties of Chl and Freshly Isolated Chloroplast at 77 K
  • b. Effects of Exogenous Tetrapyrroles on Isolated Chloroplasts

  • 3. Molecular Basis of Tetrapyrrole-Dependent Photodynamic Herbicide Selectivity

  • 4. Modulation of TDPH Activity

  • a. The Four Classes of Modulators
  • b. Response of Various Greening Groups of Plants to TDPH Modulators
  • c. Discovery of Novel TDPH Modulators

  • B. Photodynamic Insecticides

  • 1. Concept and Phenomenology

  • 2. Insecticidal Effectiveness of Ingested ALA and 1,10-Phenanthroline (Oph) or 2,2'- Dipyridyl (Dpy)

  • 3. Discovery of Other Porphyric Insecticide Modulators and Their Effects on Four Different Insect Species

  • 4. Tissue, Cellular and Subcellular Sites of Tetrapyrrole Accumulation in Various Insects

  • C. Photodynamic Cancericides

  • 1. Delta-Aminolevulinc Acid as a Photodynamic Cancer Therapeutic Agent

  • 2. Enhancement of ALA Therapeutic Effects by 1,10-Phenanthroline

  • a. Effect of ALA and 1,10-Phenanthroline on Cultured Cells
  • b. Induction of Tumor Necrosis by ALA and 1,10-Phenanthroline

  • 3. Intracellular Localization and Transport of Protoporphyrin IX in Transformed Cells

  • D. References

    XIV. Development of Photobiotechnologies

    The fundamental understanding of porphyrin and Chl chemistry and biochemistry has led to the development of several biotechnologies which are described below (Duke and Rebeiz, 1994).

    A. Photodynamic Herbicides

    1. Concept and Phenomenology

    Tetrapyrrole-dependent photodynamic herbicides (TDPH) are compounds which force green plants to accumulate undesirable amounts of metabolic intermediates of the chlorophyll (Chl) and heme metabolic pathways, namely tetrapyrroles ( Rebeiz et al, 1984; 1987; 1988; 1990; 1991; 1994; Rebeiz, 1991; Duke and Rebeiz, 1994; Reddy and Rebeiz, 1994) . In the light the accumulated tetrapyrroles photosensitize the formation of singlet oxygen which kills the treated plants by oxidation of their cellular membranes. Tetrapyrrole-dependent photodynamic herbicides usually consist of a 5-carbon amino acid, delta-aminolevulinic acid (ALA), the precursor of all tetrapyrroles in plant and animal cells, and one of several chemicals referred to as modulators. Delta-aminolevulinic acid and the modulators act in concert. The amino acid serves as a building block of tetrapyrrole accumulation, while the modulator alters quantitatively and qualitatively the pattern of tetrapyrrole accumulation (Amindari et al, 1995). The tetrapyrrole-dependent connotation is meant to differentiate between this class of photodynamic herbicides and other light activated herbicides such as paraquat which are not dependent on tetrapyrrole metabolism for herbicidal activity. During the past 13 years, the scope of TDPH research has expanded considerably, as some established herbicides and a plethora of new compounds which act via the TDPH phenomenon have been discovered.

    2. Photodynamic Effects of Metabolic Tetrapyrroles on Isolated Chloroplasts

    While delta-aminolevulinic acid (ALA)-dependent photodynamic destruction of insect and animal tissues is mainly photosensitized by protoporphyrin IX (Proto), additional Mg-containing tetrapyrroles are involved in the photodynamic destruction of plant tissues. To gain better understanding of the destructive photodynamic effects of these plant tetrapyrroles, the effects of divinyl (DV) Proto, DV Mg-Proto and its monomethyl ester and DV and monovinyl (MV) protochlorophyllides (Pchlides) on isolated chloroplasts was compared. Incubation of isolated cucumber chloroplasts with the tetrapyrroles, in the light, exhibited different effects on the pigments and pigment-protein complexes of the plastids which are described below. The state of pigment-protein complexes was monitored by analysis of pigment content and by spectrofluorometry of isolated chloroplasts af 77 K (Amindari et al, 1995).
    a. Fluorescence Properties of Chl and Freshly Isolated Chloroplast at 77 K.
    Fluorescence spectroscopy at 77 K has been used extensively to probe the effects of various metabolic tetrapyrroles on the state of organization of the chloroplast membranes. It was deemed important therefore, to discuss the 77 K fluorescence properties of freshly isolated chloroplasts before proceeding with a discussion of experimental results.

    Most of the light energy absorbed by Chls dissolved in organic solvents, is dissipated as fluorescence. At the temperature of liquid N2 (77 K) MV Chl a dissolved in diethyl ether coordinates to two solvent molecules (i. e the central Mg atom becomes hexacoordinated by axial coordination to two lewis bases) (Rebeiz and Belanger, 1984; Belanger and Rebeiz, 1984). It exhibits a major red emission maximum at 674 nm [Qy (0'-0) transition], a minor maximum at 725 nm [Qy (0'-1) transition], and Soret excitation maxima at 447 nm [By, Bx (0-0') transition] (Belanger and Rebeiz, 1984; Belanger et al, 1982), 422 nm (h1 transition) and 400 nm (h2 transition) (Weiss, 1975, 1978). Under the same conditions, MV Chl b is also hexacoordinated and exhibits a major red emission maximum at 659 nm [Qy (0'-0) transition], a minor maximum at 722 nm [Qy (0'-1) transition], and Soret excitation maxima at 475 nm [By (0-0') transition], 449 nm (h1 transition) and 427 nm (h2 transition) (Duggan and Rebeiz, 1982). The eta (h) transitions are forbidden in unsubstituted porphyrins, but become allowed in reduced porphyrins or when there is a conjugated carbonyl substituent as in the Chls (Weiss, 1978).

    In the chloroplast, MV Chl a and b are non-covalently associated with various thylakoid polypeptides. This special pigment-protein environment changes drastically the population and energy levels of various electronic transitions and results in different spectroscopic properties than in ether. As a consequence the spectroscopic properties of a given Chl-polypeptide complex, depends on the specific Chl-protein interactions within the complex. This picture is complicated further by the fact that not all Chl-protein complexes are capable of fluorescence. Depending on the structural proximity of various complexes, some Chl-polypeptides transfer their excitation energy to other fluorescing complexes, instead of emitting their excitation energy as fluorescence. These non-fluorescing Chl-polypeptides may become fluorescent only when their structural relationship to other Chl-poplypeptides is disrupted. For example Chl b does not fluoresce in healthy thylakoid membranes because it transfers its excitation energy to Chl a. It becomes fluorescent when its structural organization is disrupted.

    A fraction of the light energy absorbed by chloroplast membranes is converted to chemical energy via the process of photosynthesis. Another fraction of that energy is dissipated via several mechanisms including fluorescence. At 77 K, freshly isolated chloroplasts exhibit a deceptively simple three banded fluorescence emission spectrum with emission maxima at 683-686 nm (F686), 693-696 nm (F696) and 735-740 nm (F740) (Bassi et al, 1990; Butler and Kilajima, 1975). It is believed that the fluorescence emitted at F686 nm arises from the Chl a of the light-harvesting Chl-protein complexes (LHCII and LHCI-680), that emitted at F696 nm originates mainly from the Photosystem (PS) II antenna Chl a (CP47 and/or CP29), and that emitted at F740 nm originates primarily from the PS I antenna Chl a (LHCI-730) (Bassi et al, 1990; Butler and Kilajima, 1975). Under the same experimental conditions, each fluorescence excitation spectrum recorded at emission wavelengths of 685 (LHCII and LHCI-680), 695 (CP47 and/or CP29) or 740 nm (LHCI-730) exhibits four excitation bands with maxima at 415-417, 440 nm, 475 nm and 485 nm. The excitation band with a maximum at 415-417 nm is probably caused by the h1 transition of Chl a, while the 440 nm band corresponds to the bulk of light absorption by Chl a in the Soret region. The excitation bands with maxima at 475 and 485 nm are excitation energy transfer bands and correspond to light absorbed by Chl b and carotenoids in the Soret region. In healthy chloroplasts the photons absorbed at these wavelength by Chl b and by carotenoids, are transferred to Chl a where they are converted to chemical energy or wasted as Chl a fluorescence.

    As mentioned above, this simple picture of the fluorescence properties of thylakoid membranes is rather deceptive, since thylakoid membranes contain several Chl a and b-binding polypeptides which may not fluorescence until their structural organization is disrupted. In this context, the ratio of emission at 739-740 nm relative to that at 685 nm (F740/F686), as well as F740/F696 have been used to determine changes in the relative distribution of excitation energy between PSI and PSII which is mediated mainly by LHCII (Hipkins and Baker, 1986). The magnitude and blue shift of these fluorescence ratios have also been used to study the onset of chloroplast degradation which disrupts the normal distribution of excitation energy between the photosystems and results in a steady decrease in the F740/F696 and F740/F686 fluorescence emission ratios (Rebeiz and Bazzaz, 1978). Furthermore disorganization of the chloroplast structure results in a blue shift of the emission and excitation maxima to shorter wavelength and eventual disappearance of the emission peaks between 680 and 740 nm, and the excitation bands between 470 and 490 nm.
    b. Effects of Exogenous Tetrapyrroles on Isolated Chloroplasts
    Only one of the five exogenous tetrapyrroles failed to trigger chloroplast destruction in the light, namely divinyl DV Mg-Proto. Esterification of DV Mg-Proto to yield DV Mg-Proto monomethyl ester (Mpe) rendered this tetrapyrrole extremely destructive. While overall destructive effects were manifested by Chl a and b disappearance and the appearance of Chl degradation products, such as chlorophyllide a, and b and pheophytin and pheophorbide a, more specific effects on the pigment-protein complexes became evident from in organello 77 K fluorescence spectroscopy. DV Proto, an early intermediate in Chl a biosynthesis, affected the photosystem (PS) II antenna Chl a pigment-protein complexes, but had no effect on the PS I antenna complex and the Chl a/b light harvesting antenna complex (LHCII). On the other hand DV Mpe and DV Pchlide a, destroyed completely all the thylakoid pigment-protein complexes. As for DV-Pchlide a, it exhibited its strongest effect on the disorganization of the PS I antenna LHCI-730 complex. Altogether these results indicate that individual tetrapyrroles have distinct and different disruptive effects on the structure of thylakoid membranes in the light. Specific effects appear to be related to the position of particular tetrapyrrole in the Chl a biosynthetic chain and its electrostatic properties (Amindari et al, 1995).

    3. Molecular Basis of Tetrapyrrole-Dependent Photodynamic Herbicide Selectivity

    Originally photodynamic herbicides were assumed to be non-selective in their mode of action. Further experimentation under controlled laboratory and field conditions indicated that various ALA and modulator combinations exhibited a significant degree of photodynamic herbicidal selectivity. This selectivity appeared to be rooted (a) in the different tetrapyrrole accumulating capabilities of various plant tissues, (b) in the differential susceptibility of various greening groups (see Botanical Fallouts) of plants to the accumulation of various divinyl (DV) and monovinyl (MV) tetrapyrroles, and (c) in the differential response of various greening groups of plants to photodynamic herbicide modulators.

    4. Modulation of TDPH Activity

     The dependence of TDPH susceptibility upon the greening group affiliation of treated plants as well as upon the nature of accumulated tetrapyrroles suggested that it may be possible to chemically modulate the activity of TDPH. It was conjectured that this may be achieved with the use of chemicals that may modulate the Chl a biosynthetic pathway by forcing ALA-treated plants belonging to certain greening groups to accumulate the" wrong" type of MV or DV tetrapyrrole, while inducing other plant species belonging to other greening groups to accumulate the "right" type of MV or DV tetrapyrrole. An initial search led to the identification of 14 chemicals which acted in concert with ALA and which exhibited a definite modulating propensity toward the Chl a biosynthetic pathway. These chemicals were therefore designated as TDPH modulators. They were classified into four groups depending on their effects on the Chl a biosynthetic pathway.
    a. The Four Classes of Modulators
    In order to determine whether a compound acts as a tetrapyrrole-dependent photodynamic herbicide modulator, the chemical is usually sprayed on a plant with and without ALA, and the treated plant is kept in darkness for several hours during which tetrapyrrole accumulation takes place. After dark incubation and prior to light exposure, the plant tissues are analyzed for tetrapyrrole content. Upon exposure to light, tissues that have accumulated tetrapyrroles in darkness, exhibit rapid photodynamic damage within the first hour of illumination. The classification of a modulator as an enhancer, inducer or inhibitor of tetrapyrrole accumulation is then determined from the pattern of tetrapyrrole accumulation in the presence and absence of ALA and modulators.

    Based on their mechanism of action TDPH modulators have been classified into four distinct groups: (a) enhancers of ALA conversion to divinyl protochlorophyllide (DV Pchlide a), which enhance the conversion of exogenous ALA to DV Pchlide a, (b) enhancers of ALA conversion to MV Pchlide a, which enhance the conversion of exogenous ALA to MV Pchlide a, (c) inducers of tetrapyrrole accumulation, which induce the plant tissues to form large amounts of tetrapyrroles in the absence of exogenously added ALA, and (d) inhibitors of MV Pchlide a accumulation, which appear to block the detoxification of DV tetrapyrroles by inhibiting their conversion to MV tetrapyrroles. Of all the aforementioned modulators, only inducers of tetrapyrrole accumulation are capable of causing tetrapyrrole accumulation in the absence of added ALA. The three other classes of modulators do not lead to significant levels of tetrapyrrole accumulation in the absence of added ALA. In all cases, the use of ALA together with a modulator results in enhanced tetrapyrrole accumulation and photodynamic damage over and beyond the levels caused by ALA alone.
    b. Response of Various Greening Groups of Plants to TDPH Modulators
    Mode of action determinations were performed on representative plant species belonging to the three known greening groups of plants, namely: cucumber, soybean and Johnsongrass, The results indicated that (a) a modulator that acts in a certain way on the Chl biosynthetic pathway of one greening group of plants does not necessarily act the same way on plant species belonging to a different greening group, (b) different plant species belonging to the same greening group tend to exhibit similar Chl a biosynthetic reactivities toward a given modulator and (c) modulators that belong to the same chemical category tend to exhibit the same Chl a biosynthetic modulating activity toward a particular plant species. Altogether the above results suggest that it is possible to make certain predictions about the mode of action of a modulator toward a particular plant species belonging to a particular greening group, once the mode of action of the chemical category to which the modulator belongs, has been determined for that particular greening group.
    c. Discovery of Novel TDPH Modulators
    Because of the central importance of modulators to the performance of TDPH, considerable time and efforts have been devoted during the past several years, to the discovery of novel TDPH modulators. The experimental strategy used in that successful undertaking used two dimensional and three dimensional computer modeling and resulted in the discovery of several hundred potent TDPH modulators (Rebeiz et al, 1990; 1991; 1994; Reddy and Rebeiz, 1994).

    B. Porphyric Insecticides

    1. Concept and Phenomenology

    The discovery of porphyric insecticides (Rebeiz et al, 1988; 1990; 1995; Rebeiz, 1993; Gut et al, 1993; 1994a; 1994b) was a direct fallout of the discovery and development of photodynamic herbicides . Since plant and animal cells share the same tetrapyrrole biosynthetic pathway from ALA to protoporphyrin IX (Proto), it was conjectured that it should be possible to adapt the TDPH phenomenon to the photodynamic control of insects. Initial trials were performed on Trichoplusia ni (cabbage looper) larvae. Demonstration of the potential for tetrapyrrole accumulation in insects was initially achieved by spraying T. ni larvae with ALA (40 mM) + 2,2,-dipyridyl (Dpy) (30 mM) (Rebeiz et al, 1988). Treated larvae were placed overnight in darkness at 28øC in order to allow for putative tetrapyrrole accumulation. Extraction of treated, dark-incubated larvae with ammoniacal acetone, followed by spectrofluorometric examination of the larval extract, revealed the accumulation of massive amounts of a fluorescent compound which was not present in control larvae sprayed with solvent only. Following chemical derivatization coupled to spectrofluorometric analysis, the accumulated compound was identified as a tetrapyrrole, specifically Protoporphyrin IX (Proto). A high degree of correlation was observed between Proto accumulation in darkness and larval death in the light. A few hours after exposure to light, the larvae became sluggish and flaccid due to loss of body fluids. Death was accompanied by extensive desiccation

    2. Insecticidal Effectiveness of Ingested ALA and 1,10-Phenanthroline (Oph) or 2,2'-Dipyridyl (Dpy)

    Since control of insects by ingestion is as viable an option as control by spraying, and offers certain advantages under household conditions, studies were conducted to determine whether combinations of ALA and porphyric insecticide modulators would be effective if ingested with the food (Rebeiz et al, 1990). Initially the effect of ALA (16 mM final concentration) and Oph (12 mM final concentration) were determined by incorporating them into the diet of T. ni. larvae. Upon exposure to light, following 17 hr of dark incubation, larvae underwent violent convulsions and vomiting and died within 20-40 seconds. Tetrapyrrole analysis of the treated larvae immediately after dark incubation revealed significant amounts of Proto and Zn-Proto accumulation. Correlation between tetrapyrrole accumulation and larval death was significant Similar results were obtained when ALA and DPY were administered to the larvae with the diet. The above results indicated that in addition to contact via spraying, porphyric insecticides had the potential to be very potent when ingested.

    3. Discovery of Other Porphyric Insecticide Modulators and their Effects on Four Different Insect Species

    Structure-function photodynamic herbicidal studies (Gut et al, 1994b; Rebeiz et al, 1995) have led to the assembly of two databases of commercially available compounds with potential photodynamic herbicidal properties. The databases consisted of a set of 6-membered N-heterocyclic compounds, and a set of 5-membered N-heterocyclics. A substructure computer search of these databases identified 322 putative photodynamic herbicide modulators. Extensive testing of these modulators on a variety of plant species led to the identification of about 150 modulators with excellent photodynamic herbicidal properties. Encouraged by these results, a screening effort was undertaken to determine whether these 150 modulators exhibited porphyric insecticidal properties. Screening by food ingestion was performed on the german cockroach, cotton boll weevil, corn earworm and cabbage looper as described below. Thirty six compounds belonging to 10 different chemical families (templates) were effective (> 70 % mortality) against at least one insect species. Of the 36 modulators, 10 modulators exhibited potent activity toward cockroaches. One additional modulator, namely 1-phenyl pyrrole, exhibited considerable activity against cockroaches under closed, non aerated conditions.

    4. Tissue, Cellular and Subcellular Sites of Tetrapyrrole Accumulation in Various Insects

    For a more thorough understanding of the mode of action of porphyric insecticides, the phenomenology of tissue, cellular and subcellular sites of tetrapyrrole accumulation in representative insect species was investigated (Lee and Rebeiz, 1995). In T. ni larvae sprayed with ALA (40 mM) + Dpy (30 mM), the integument, hemolymph and gut of sprayed early fifth instar larvae were separated and analyzed for pigment content. On a unit protein basis, about 59% of the accumulated Proto was observed in the hemolymph, 35% in the gut and 6% in the integument.

    Further understanding of the response of insect organs and tissues to porphyric insecticide treatment was obtained by investigating the response of isolated organs and tissues to incubation with ALA + Dpy or ALA + Oph (Lee and Rebeiz, 1995). In these experiments, the following insects were used: Adult Blattella germanica (german cockroach), Adult Anthonomus grandis (cotton boll weevil), fifth instar larvae of Heliothus zea (corn earworm) and fifth instar larvae of T. ni (cabbage looper). In T. ni, and H. zea, significant Proto accumulation was observed in incubated midgut, and fat bodies. Proto accumulation occurred when tissues were incubated with Dpy, ALA + Dpy, Oph, and ALA + Oph. No response to treatment with ALA alone was observed. In cockroaches, more of the Proto appeared to accumulate in the male and female guts than in their abdomen. As in T. ni and H. zea, the response was elicited by each of the treatments that included Dpy or Oph. Cotton boll weevil abdomens appeared to be less responsive than the abdomens of the other three species.

    To determine whether Proto accumulation resulted in photodynamic damage of incubated tissues, T. ni midguts were incubated in darkness either in buffer, with ALA, or with Oph + ALA. Oxygen consumption of the tissue was then monitored before and after exposure to 2-hr of illumination. It was assumed that decrease in O2 consumption indicated photodynamic damage and cell death. A thirty percent decrease in O2 consumption was observed in mid guts treated with Oph or with ALA + Oph after 2-hr in the light (Lee and Rebeiz, 1995). The decrease in oxygen consumption observed in isolated T. ni midguts suggested that toxicity of porphyric insecticides may result, among other things, from photodynamic damage to mitochondria. This issue was next investigated. Fifth-instar T. ni larvae were placed on diets containing ALA (4 mM) and Oph (3 mM) in darkness for 17-hr. After dark-incubation, the site of Proto accumulation in various subcellular components of the larvae was determined. Most of the Proto was found in the mitochondrial (37%) and microsomal (35%) fractions, while the balance (28%) was found in the cytosol.

    To determine the possible photodynamic effects of mitochondrial Proto accumulation upon mitochondrial function, mitochondria were isolated from fifth-instar T. ni larvae which were dark-treated for 17-hr with ALA (4 mM) and Oph (3 mM) (Lee and Rebeiz, 1995). The isolated mitochondrial suspension was exposed to 900 W.m-2 of white fluorescent light for 30 min at 25øC before monitoring the activity of various mitochondrial marker enzymes, namely: succinate oxidase, NADH dehydrogenase and NADH-cytochrome c reductase. ALA + Oph treatment exhibited deleterious effects on mitochondrial activities before illumination, which tended to obscure the possible involvement of singlet oxygen in enzyme Photoinactivation. However, the rate of NADH-cytochrome c reductase activity, appeared to decline more rapidly in the light, in Proto-enriched than in control mitochondria. This in turn suggested the possible involvement of singlet oxygen in the enhanced inactivation of mitochondrial cytochrome c reductase activity by light.

    C. Photodynamic Cancericides

    1. Delta-Aminolevulinc Acid as a Photodynamic Cancer Therapeutic Agent

    Following in the footsteps of the photodynamic herbicide and porphyric insecticide technologies, the porphyrin-inducing properties of delta-aminolevulinic acid (ALA) have been adapted for the photodynamic destruction of cancer cells (Kennedy et al, 1990; Peng et al, 1992; Wolf et al, 1993; Grant et al, 1993; Cairnduff et al, 1994; Svanberg et al, 1994). Topical administration ALA to various skin lesions has, in particular, been very successful in clinical trials. For instance Kennedy et al (1990) treated successfully basal cell carcinomas, superficial squamous cell carcinomas, and actinic keratoses, wit a response rate of 90 % for basal cell carcinomas. Overall, topical, oral, or systemic administration of ALA and subsequent photodynamic therapy has been successful in a variety of tumor models, including amelanotic melanomas (Abels et al, 1994), pancreatic cancer (Regula et al, 1994), and colon tumors (Orth et al, 1994).

    The successful use of ALA as a photodynamic cancer therapeutic agent is largely due to the unique properties of this 5-carbon amino acid (Kennedy et al, 1994). ALA is a small water soluble molecule. It can easily penetrate cells (Sziemes et al, 1994; Van der Veen et al, 1994), and is easily taken up by transformed cells (Divaris et al, 1990; Peng et al, 1987; Bedwell et al, 1992). Furthermore photoactivation of ALA-induced Proto requires less light energy than commonly used photosensitizing drugs (Kennedy et al, 1992), and ALA and Proto are rapidly cleared from the circulation within 16-48 hour after treatment (Bedwell et al, 1992; Kennedy and Pottier, 1992; Pottier et al, 1986).

    2. Enhancement of ALA Therapeutic Effects by 1,10-Phenanthroline

    It has been demonstrated that Proto accumulation and concommittant transformed cell (Rebeiz et al, 1992; 1994) and tumor (1996a) destruction are enhanced considerably by addition of the tetrapyrrole modulator 1,10-phenanthroline (Oph) to ALA formulations.
    a. Effect of ALA and 1,10-Phenanthroline on Cultured Cells

    Proto accumulation in rapidly proliferating mammalian cells [gibbon monkey lymphpma cell line MLA 144, human myelogenous leukemia cell line K562, murine methyl-cholanthrene induced sarcoma cells (Meth-A), and the murine fibrosarcoma cell line WEHI 164 clone 13] was induced by treatment with 1.0 mM ALA. In darkness, significant Proto accumulation became evident within 3.5 hours of incubation. In the light, the accumulated tetrapyrroles triggered destruction of the treated cells within the first 30 minutes of illumination. Protoporphyrin IX accumulation and specific cell lysis increased significantly by inclusion of 0.75 mM 1,10-phenanthroline (Oph), a tetrapyrrole biosynthesis modulator (Rebeiz et al, 1992, 1994). Slower growing untransformed cells did not accumulate significant amounts of Proto following ALA and Oph treatment unless stimulated to proliferate with the mitogenic lectin Concanavalin A.
    b. Induction of Tumor Necrosis by ALA and 1,10-Phenanthroline
    Solid Meth-A tumors grown in syngeneic BALB/c mice accumulated significant amounts of Proto 3 hr after in vivo treatment with ALA. 1,10-Phenanthroline synergized with ALA and enahnced significantly the induction of Proto in the tumors. ALA and Oph-based phototreatment of mice bearing the Meth-A solid tumors resulted in tumor necrosis as determined by significant reduction in both size and histopathology, with little damage to surrounding normal tissues (Rebeiz et al, 1996a).

    3. Intracellular Localization and Transport of Protoporphyrin IX in Transformed Cells

    Although porphyrin biosynthesis and accumulation has been investigated in normal cells, little is known about the intracellular localization and transport of porphyrins in transformed cells. It has been proposed that the biosynthesis of Proto in normal animal cells requires the cooperation of cytoplasm and mitochondria (Granick, 1963). The formation of ALA, the first committed step in the porphyrin biosynthetic pathway has been shown to occur in the mitochondria (Shemin et al, 1954; Kikuchi et al, 1956, Kikushi and Mayashi, 1981). ALA is subsequently transported to the cytoplasm where it is believed to be converted to coproporphyrinogen III (Coprogen) (Granick, 1963; Granick and Mauzerall, 1958; Bograd, 1958; Frydman et al, 1978). The idea that ALA is converted to Coprogen in the cytoplasm of normal animal cells, evolved from the finding that osmotic lysis of chicken erythrocytes in distilled water released soluble enzymes that are capable of converting ALA to Coprogen (Granick, 1963). This lysing technique, however, did not differentiate between cytoplasmic and organellar soluble enzymes. More recently some of these soluble enzymes have been cloned, but little attention was paid to their intracellular localization (Jordan, 1990; 1994). It has also been demonstrated that in normal cells, Coprogen is transported into the mitochondria via an unknown mechanism, for further metabolism (Granick 1963; 1967). In the mitochondria, Coprogen oxidase and protoporphyrinogen oxidase convert Coprogen to Proto (Sano and Granick, 1961; Dailey, 1990). Proto is then converted to heme by ferrochelatase, a mitochondrial enzyme, which inserts iron into the Proto macrocycle (Goldberg et al, 1956; Dailey and Nacelles, 1974). Subsequently, Proto and heme are exported from the mitochondria to other subcellular organelles via an unknown mechanism (Wijesekera and Dolphin, 1985; Richelli et al, 1995). McEnery et al. (1992) proposed that a complex of proteins that span the inner and outer mitochondrial membranes is involved in porphyrin transport. This complex consists of three interacting subunits which form the mitochondrial peripheral-type benzodiazepine receptor (M-PBR). The endogenous ligand of M-PBR was identified as Proto (Verma et al, 1987; Verma and Snyder, 1988), and more recently, Taketani et al. (1994; 1995) have shown that Coprogen and heme bind to the M-PBR, and that M-PBR antagonists inhibit the conversion of Coprogen to Proto. Rebeiz et al (1996), have shown that in transformed cells, in the presence of added Oph: (a) the cytoplasm is the site of Coprogen formation from added ALA, (b) Coprogen can be transported to the mitochondria, where it is converted to Proto, and (c) conversion of cytoplasmic Coprogen to Proto by mitochondria is an ATP-dependent process.

    References

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