Chlorophyll Biosynthesis

18.4 Tissue Cellular and Subcellular Sites of Tetrapyrrole Accumulation. . . 425 spectrofluorometry (Rebeiz 2002). Concomitant photodynamic damage was assessed by monitoring the decrease in oxygen consumption of the incubated tissue in the light. Oxygen consumption was determined polarographically, using a Clark oxygen electrode. The results of isolated organ and tissue investigations for each of the aforementioned four insect species are summarized Table 18.10. In T. ni, and H. zea, significant Proto accumulation was observed in the midgut, and fat bodies. Proto accumulation occurred when tissues were incubated with Dpy, ALA + Dpy, Oph, and ALA + Oph (Table 18.10). 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 in 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 h of illumination. It was assumed that decrease in O2 consumption indicated photodynamic damage followed by cell death. A 30 % decrease in O2 consumption was observed in mid guts treated with Oph or with ALA + Oph after 2 h in the light (Lee and Rebeiz 1995). 18.4.3 Subcellular Localization of Proto Accumulation in T. ni The decrease in oxygen consumption observed in isolated T. ni midguts (Table 18.10), suggested that toxicity of porphyric insecticides may result, among other things, from photodynamic damage to mitochondria. This issue was investigated by Lee and Rebeiz as described below (Lee and Rebeiz 1995). Fifth-instar T. ni larvae were placed on diets containing ALA (4 mM) and Oph (3 mM) in darkness for 17 h. 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. In order to ascertain that the mitochondrial Proto was not due to contamination by microsomal and cytosolic Proto, The Proto content of mitochondria purified on Percoll gradients was also determined. Percoll- purified mitochondria were highly active as evidenced by their succinate cytochrome c reductase activity, and contained 534 nmol Proto per 100 mg mito- chondrial protein. These results suggested that Proto formation may take place in the mitochondria and microtomes both of which need Proto for heme formation, while the presence of Proto in the cytosol may be due to leakage from the mitocho- ndrial and microsomal compartments.

426 18 Porphyric Insecticides 18.4.4 Photodynamic Effects of Proto Accumulation on Mitochondrial Function in T. ni 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 h with ALA (4 mM) and Oph (3 mM). The isolated mitochondrial suspension was exposed to 900 W/m2 of white fluorescent light for 30 min at 25 C before monitoring the activity of various mitochondrial marker enzymes, namely: succinate oxidase, NADH dehydrogenase and NADH-cytochrome c reductase. All three-enzyme activities decreased significantly in a time-dependent manner in comparison to dark mitochondrial controls. These results strongly indicated that Proto accumulation in mitochondria triggers mitochondrial damage in the light and may contribute significantly to photodynamic damage in treated insects (Lee and Rebeiz 1995). 18.5 Screening of Other Porphyric Insecticide Modulators and Their Effects on Four Different Insect Species Earlier photodynamic herbicide structure-function studies described elsewhere led to the assembly of two databases of commercially available compounds with potential photodynamic herbicidal properties (Rebeiz et al. 1990b, 1991). These databases consisted of a set of 6-membered N-heterocyclic compounds (Rebeiz et al. 1991) and a set of 5-membered N-heterocyclics (Rebeiz et al. 1990b). A substructure computer search of these databases identified 322 putative photody- namic herbicide modulators (see Chap. 17). Extensive testing of these modulators on a variety of plant species led to the identification of about 150 modulators with excellent photodynamic herbicidal properties (Rebeiz et al. 1990b). 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. For T. ni and H. zea, ALA (4 mM) and a modulator (3 mM) were added to liquefied Waldbauer’s medium (Waldbauer et al. 1984) at 55–60 C. The mixture was blended for 2 min in a Sorval Omnimixer. The treated and control diets (the latter lacking ALA and modulators) were poured into 12-ml plastic molds and were allowed to cool down and to solidify before storage in a refrigerator at 4 C. The food was generally used within 2 days, and was never stored for more than 2 weeks. Treatment of T. ni consisted of placing 15–20 third-instar larvae with one block of control or treated food, in a cardboard cup (about 9 cm h  10 cm diameter) sealed with a plastic lid. Each treatment was replicated three times. Treatment of H. zea entailed placing a 3 ml control or treated diet cube and a single

18.5 Screening of Other Porphyric Insecticide Modulators and Their Effects. . . 427 third instar H. zea larvae in each cell of a 20 cell plastic tray. The tray was sealed with a glass plate to prevent the escape and/or desiccation of the larvae. For both species, treatment was replicated three times. After 17 h of dark incubation at room temperature, the control and treated insects were placed in the growth chamber at 28 C, [about 21.1 mW·cmÀ2 of white light (metal halide)] under a 14 h light-10 h dark photoperiod. After 6 h in the light, untreated diet was added to each treatment. The untreated food was replenished daily. Insect death was monitored over a 6-day period. Anthonomus grandis were obtained from a colony maintained at the Boll Weevil Research Laboratory, USDA, SEA, Mississippi State, Mississippi. A single tray of eggs dispersed on boll weevil diet was received weekly and held at room tempera- ture until adults emerged. ALA and a modulator were added to liquefied, warm, boll weevil diet (BioServe Inc.) to a final concentration of 8 mM of ALA and 6 mM modulator. The mixture was blended for 2 min in a Sorval Omnimixer. Treated and control diets (the latter lacking ALA and modulators) were poured into 12 ml plastic molds and allowed to cool down and solidify before storage in a refrigerator at 4 C. Treatment consisted of placing 15 adults and a block of control or baited food in a cardboard carton (9 cm h  10 cm diameter) sealed with a plastic lid. In order to minimize desiccation, a small petri dish (about 2.5 cm in diameter) containing cotton moistened with water was placed in the bottom of each carton. The baited food was replenished daily for the duration of the experiment. Dark incubation, exposure to light and evaluation of mortality was as described for T. ni and H. zea. Blattella germanica were obtained from a colony maintained in a Biotronette Mark III Environmental Chamber at 28 C, and 50 % relative humidity. The chamber was set for an 8-h light-16-h dark photoperiod. The colony was initiated from egg cases purchased from Carolina Biological Supply and was maintained on a diet of dog food and water. Sub-colonies were established weekly in separate 42-cm  27-cm plastic animal cages. Waldbauer’s medium was prepared exactly as described for T. ni. However concentrations of ALA and modulator were raised to 24 and 16 mM, respectively. Treatment consisted in placing a 12 ml baited block of food and 15 adults in a cardboard carton sealed with a plastic lid. As was described for A. grandis, a small petri dish containing moistened cotton was placed in the bottom of the carton. Control and treated containers were then placed in darkness at room temperature for 40 h. After dark-incubation the cartons were placed under subdued light (20–40 ft. candles) for the duration of the experiment. The baited diet, and water in the cotton dish, were replenished daily. Mortality was recorded over a 6-day period. The results of the screening effort are described in Table 18.11. Thirty six compounds belonging to ten different chemical families (templates) were effective (>70 % mortality) against at least one insect species. T. ni was generally more susceptible than the other species. Structure-activity studies of some of these compounds are described below.

Table 18.11 Porphyric Insecticide Modulators with 70 % or Better Activity Against Third Instar Larvae of T. ni, H. zea, and Against Adult A. grandis and B. germanica Mortality after 6 days (%) T. H. A. B. Template Modulator ni zea grandis germanica 1,10-Phenanthroline 1,10-Phenanthroline 100 100 90 100 87 4-Methyl-1,10-Phenanthroline 99 95 76 30 96 5-Methyl-1,10-Phenanthroline 97 55 64 93 27 4,7-Dimethyl-1,10-Phenanthroline 100 90 85 38 5,6-Dimethyl-1,10-Phenanthroline 97 50 51 82 3,4,7,8-Tetramethyl-1,10- 85 79 44 0 100 Phenanthroline 7 5-Nitro-1,10-Phenanthroline 99 63 58 0 67 5-Chloro-1,10-Phenanthroline 96 84 40 53 (Di)pyridyls 4,7-Diphenyl-1,10-Phenanthroline 96 0 0 7 2,20-Dipyridyl 100 100 100 0 4,40-Dimethyl-2,20-Dipyridyl 71 25 20 0 Phenyl-2-Pyridyl ketoxime 71 5 7 2,20-Dithiobis (pyridine N-oxide) 73 0 67 0 100 Benzyl viologen dichloride 0 47 100 80 monohydrate 0 93 Quinoxaline Neutral red 81 20 11 40 8-Hydroxyquinoline 8-Hydroxy-7-(4-sulfo-1- 74 35 2 83 13 naphtylazo)-5-quinoline 80 8-Hydroxy-5-sulfonic acid 86 10 0 3 monohydrate 0 27 8-Hydroxy-7-iodo-5-quinoline 78 10 0 0 2-Oxypyridine 2-Metoxy-5-nitropyridine 56 0 64 3 Isocarbostyril 84 0 60 3 100 Pyridiniums 1-1-Diethyl-4,4-carbocyanine iodide 67 95 100 100 100 1-1-Diethyl-2,4-cyanine iodide 36 11 53 100 Cetylpyridinium chloride 49 0 78 monohydrate iodide 2-Chloro-1-methylpyridinium iodide 49 5 0 2-(4-(Dimethylamino)-styril)- 30 10 89 ethylpyridinium 2-(4-(Dimethylamino)-styril)- 11 0 73 methylpyridinium Poly (4-vinylpyridinium) dichromate 85 5 4 5-Phenyl-2-(4-pyridyl) oxazole 76 20 0 Bis-N-methyl acidinium nitrate 75 0 56 Pyrrole 3-Ethyl-2-methyl-4,5,6,7- 93 25 11 tetrahydroindol-4-one Tert-butyl-4-acetyl-3,5-dimethyl- 71 0 9 2-pyrrole-carboxylate Pyrrolidine 1-Phenylpyrrole 71 0 9 Tetrazole –b – – 4-Pyrollidinopyridine 3-30-(4,40-Biphenylene) bis ––– Thiazole 3,6-Dimethyl-2- ––– (4-dimethylaminophenyl) Thioflavin T ––– Adapted from Gut and Rebeiz, unpublished aProcedural details are given in the text bNot determined

18.6 Structure-Activity Studies of Porphyric Insecticide Modulators 429 18.6 Structure-Activity Studies of Porphyric Insecticide Modulators The identification of a significant number of chemicals exhibiting insecticidal activity encouraged detailed structure-function analyses of these modulators. Accordingly structure-function studies of modulators belonging to the Oph, Dpy, pyridinium, oxypyridine, pyrrole and 8-hydroxyquinoline templates were investigated. The chem- ical structures of the selected modulators and their template affiliation are depicted below in Figs. 18.5, 18.6 and 18.7. Quantitative structure activity relationship (QSAR) analysis was performed on a Digital Equipment Co. workstation Model 3520, operating on a VMS platform. Chem-X software, (Chemical Design Limited, Oxford, England) was used to build 3-dimensional chemical structures and to carry out QSAR analysis. The latter involved, among other things: (a) optimization of the chemical structures via Mopac (QCPE, version 5, 1989) using MNDO or PM1 Hamiltonians, (b) writing the optimized structures of Oph and its analog to a database, (c) calculation of 24 different electronic and physical organic properties for each structure, i.e. descriptors, (d) correlation analysis between the 24 descriptors and biological activity, and (e) stepwise multiple regression analysis to determine the nature of relationships between biological activity and various descriptors. Calculation of electrostatic potential energy levels at various sites of a given molecule was performed using Chem-X software. Chem-X treats the charge on each atom in a molecule as a point charge positioned at the center of the atom. A positive unit charge equivalent to that of a proton is placed at each grid point and the electrostatic interaction between groups of atoms and the unit charge is calculated. The number of grid points used in the calculation, one point per Angstrom in this case, is usually set by the operator. After calculations were completed, electrostatic isopotential contour lines were drawn. The level of potential energy in kcal per mole was also selected by the operator. We chose values of 10 kcal/mol for positive potential energy levels and À10 kcal/mol for negative potential energy levels. Since the interaction of a positive probe with a positive region of the molecule generated positive energy levels they were interpreted as repelling. Likewise since the inter- action of a positive charge probe with a negative region of the molecule generated negative energy levels they were interpreted as binding or attracting energy levels. In this manner the attraction or repulsion at various loci of a particular molecule toward a positive charge was well defined by the negative and positive potential energy contour lines respectively, which in fact delineated positive charge binding or repelling electrostatic volumes surrounding various sections of a molecule. Quantitative and positional differences between the electrostatic fields of various modulators were calculated by determining the exclusive positive charge binding and repelling volumes for each analog in comparison to a reference molecule. The calculation of exclusive volumes (i.e. non overlapping volumes between any two molecules) was achieved via a Chem-X software module that calculated exclusive electrostatic field volumes for pairs of molecules from the electrostatic volumes of each individual molecule. In so doing it became possible to compare quantitative and qualitative positional differences between various analogs within each template.

430 18 Porphyric Insecticides Fig. 18.5 Chemical names and structures of the substituted phenanthrolines used in the structure- activity investigations

18.6 Structure-Activity Studies of Porphyric Insecticide Modulators 431 Fig. 18.6 Chemical names and structures of the substituted pyridyls used in the structure-activity investigations

Fig. 18.7 Chemical names and structures of substituted pyridiniums, oxypyridines, quinolines and pyrroles used in the structure-activity investigations

18.6 Structure-Activity Studies of Porphyric Insecticide Modulators 433 In the study of structure/function correlations the emphasis was placed on the analysis of relationships between tetrapyrrole modulating activity, photodynamic damage and alterations in the electrostatic field of various modulators. With this approach the underlying hypothesis was structural complementarity between enzy- matic receptor sites and modulators that favored electrostatic binding of various modulators to or close to the receptor sites of enzymes that catalyze various reactions of the heme biosynthetic pathway. It was conjectured that the detection of exclusive positive or negative charge binding volumes in groups of analogs that exhibited similar tetrapyrrole biosynthesis modulating activity may be an indication that these analogs may exert their effects by binding to the same enzymatic receptor site. In all cases the modulation of tetrapyrrole biosynthetic activity by various analogs was related to the presence of unique positive charge binding or repelling volumes. This in turn suggested that various modulators exerted their effects by binding to specific negatively charged areas that may well be close to tetrapyrrole biosynthetic enzyme reaction sites. Early fourth-instar T. ni larvae were used in evaluating modulator effects on Proto accumulation and larval mortality. Chemicals were incorporated into liquefied Waldbauer’s medium as described in section above in “IV”. Twenty six larvae were placed in each diet cup and allowed to feed in darkness for 17 h. Following dark incubation, six larvae were assayed for tetrapyrrole accumulation. Mortality of the remaining 20 larvae was monitored prior to light exposure and daily thereafter, for a period of 6 days. Modulators were evaluated alone and in combination with ALA in a randomized complete block design with three replicates. Percent larval mortality was arcsine transformed prior to statistical analysis. Structure-Activity relationship of five selected modulators are reported below. 18.6.1 Structure-Activity Relationship of Substituted Phenanthrolines The porphyric insecticide modulating activity of Oph and eight of its analogs are depicted in Table 18.12. The insecticidal efficacy of Oph and its analogs was closely associated with their ability to enhance the conversion of exogenous ALA to Proto (Table 18.13). As was observed for photodynamic herbicidal effects in plants, (Rebeiz et al. 1990b) the presence of N atoms at position 1 and 10 of the macrocycle was essential for porphyric insecticidal activity. This was evidenced by the very limited activity of phenanthrene, in which position 1 and 10 are occupied by carbon instead of N atoms (Fig. 18.5, Table 18.12, # 7, 8). On the other hand, enhancement of Proto formation and porphyric insecticidal activity were main- tained following peripheral methyl, chloro and nitro group substitution of Oph (Table 18.12, # 11–18). In contrast, enhancement of Proto formation and photody- namic toxicity was diminished by phenyl or benzyl (data not shown) substitution (Table 18.12, # 5, 6, 9, 10).

434 18 Porphyric Insecticides Table 18.12 Effects of ALA, phenanthrene and 1,10-phenanthroline (Oph) on Proto accumulation and larval death in T. ni Proto content Larval mortality after 6 days Entry Treatmenta (nmol/100 mg protein) in the greenhouse (%) 1 Control 1.5ab 0.0a 2 ALA 3.0a 15.0abcd 3 2,9-Dimethyl-4,7-diphenyl Oph 0.8a 0.0a 4 Above + ALA 1.6a 10.0abc 5 2,9-Dimethyl-4,7-diphenyl Oph 1.2a 0.0a 6 Above + ALA 1.6a 18.3abcd 7 Phenanthrene 1.0a 0.0a 8 Above + ALA 3.9a 23.3bcd 9 4,7-Diphenyl Oph 1.3a 0.0a 10 Above + ALA 8.6a 36.7 cd 11 3,4,7,8-Tetramethyl Oph 5.1a 11.7abcd 12 Above + ALA 117.0c 95.0e 13 4,7-Dimethyl Oph 15.9a 8.3ab 14 Above + ALA 131.5c 96.7e 15 5-Cl-Oph 2.0a 10.0abc 16 Above + ALA 133.9c 96.7e 17 5-Nitro-Oph 50.4b 41.7d 18 Above + ALA 190.1d 98.3e 19 Oph 8.6a 5.0a 20 Above + ALA 207.0d 100.0e Correlation coefficient 0.88 Level of significance 0.1 % Adapted from Gut et al. (1993) aTreatment consisted of control diet lacking any added ALA or modulator, diet containing 4 mM ALA, 3 mM modulator or 4 mM ALA + 3 mM modulator, in a randomized complete block design with three replicates. Percent larval death was arcsine transformed prior to statistical analysis bMeans followed by the same letter within a column are not significantly different at the 5 % level of significance Quantitative structure activity calculations suggested a relationship between peripheral group substitution and some physico-chemical properties of the substituted compounds. Electron density changes in Oph and its analogs that appeared to be related to reduced efficacy included (Gut et al. 1993) (a) Appearance of positive charge binding volumes at position 4 and 7 of the 1,10-phenanthroline macrocycle, which flanks positive charge repelling volumes, (b) a dramatic increase in super- delocalisability (i.e. electron density) over some unoccupied molecular orbitals, and (c) electronic charge at position 1 and 10 of the macrocycle. Large increases in Van der Waals volumes also exerted negative effects on insecticidal efficacy (Gut et al. 1993). 18.6.2 Structure-Activity Relationship of Substituted Pyridyls The porphyric insecticide modulating activity of Dpy and eight of its analogs are depicted below in Table 18.13. The insecticidal efficacy of Dpy and its analogs was

18.6 Structure-Activity Studies of Porphyric Insecticide Modulators 435 Table 18.13 Effects of ALA and substituted dipyridyls, on Proto accumulation and larval mortality in T. ni Proto content Larval mortality after 6 days Entry Treatmenta (nmol/100 mg protein) in the greenhouse (%) 1 Control 0.9ab 0.0a 2 ALA 2.3a 1.7a 3 2,20-Dipyridyl disulfide 1.0a 0.0a 4 Above + ALA 1.6a 0.0a 5 2,20-Biquinoline 0.9a 0.0a 6 Above + ALA 2.0a 0.0a 7 4,40-Diphenyl-2,20-dipyridyl 1.2a 1.7a 8 Above + ALA 2.3a 1.7a 0.0a 9 2,20-Dithiobis (5-nitropyridine) 0.7a 10 Above + ALA 1.8a 5.0a 0.0a 11 2,20-Dithiobis (pyridine) N-oxide 0.7a 12 Above + ALA 10.46a 56.7c 13 4,40-Dimethyl-2,20-dipyridyl 1.9a 0.0a 14 Above + ALA 18.1a 28.3b 15 Phenyl 2-pyridyl ketoxime 1.5a 1.7a 16 Above + ALA 21.9a 33.3b 17 2,20:60,200-Terpyridine 6.2a 11.7a 18 Above + ALA 181.1b 98.2d 19 2,20-Dipyridyl 15.0a 48.3c 20 Above + ALA 176.3b 100.0d Correlation coefficient 0.91 Level of significance 0.1 % Adapted from Gut et al. (1993) aTreatment consisted of control diet lacking any added ALA or modulator, diet containing 4 mM ALA, 3 mM modulator or 4 mM ALA + 3 mM modulator, in a randomized complete block design with three replicates. Percent larval death was arcsine transformed prior to statistical analysis bMeans followed by the same letter within a column are not significantly different at the 5 % level of significance closely associated with their ability to enhance the conversion of exogenous ALA to Proto (Table 18.13). As was observed with Oph, the presence of N atoms at position 1 and 10 of the macrocycle appeared to be essential for porphyric insecticide activity. This was evidenced by a 30 % reduction in activity in phenyl 2-pyridyl ketoxime with a N atom at position 1 and a carbon atom at position 10 (Fig. 18.7) as compared to dipyridyl ketoxime with 2 N atoms at positions 1 and 10 of the macrocycle (data not shown). The presence of a third 6-membered N heterocyclic ring as in 2,20:60200-terpyridine did not decrease insecticidal activity (Fig. 18.6, Table 18.14, # 17). Ring substitution at the periphery of the Dpy macrocycle, however, had a highly negative effect on insecticidal performance. For example the addition of phenyl, benzyl, or methyl groups as in 4,40-diphenyl-Dpy, biquinoline and 4,40-dimethyl-Dpy, dramatically reduced the insecticidal activity (Table 18.13, # 5, 6, 7, 8, 13, 14).

436 18 Porphyric Insecticides Quantitative structure activity calculations again revealed a potential relationship between some physico-chemical properties of various analogs and photodynamic insecticidal activity. For example, substantial increase in the distance between N1 and N10 which ensued following the insertion of a disulfide bridge between the two rings, as in 2,20-dipyridyl disulfide, eliminated completely the insecticidal activity (Fig. 18.6, Table 18.14, # 3, 4). As was observed for Oph analogs, electron density changes in Dpy and its analogs that appeared to be related to reduced efficacy included, (a) appearance of positive charge binding volumes at position 4 and 40 of the dipyridyl macrocycle, which flanked positive charge repelling volumes, (b) the acquisition of a fourth phenyl or pyridyl ring which generated alternating positive charge binding and repelling electrostatic fields between rings C and D, and (c) a change in sign and magnitude of the 1, 2, 20 10 torsion angles which was accompanied by the appearance of positive charge repelling volumes at the C50-C60, or C60 positions (Gut et al. 1993). 18.6.3 Structure-Activity Relationship of Substituted Pyridiniums The structure-activity relationships of four substituted Pyridiniums was investigated (Fig. 18.4). Porphyric insecticide damage was positively correlated with Proto accumulation except for 1,10-diethyl-4,40-carbocyanine iodide (Table 18.14, # 9,10). Although light was absolutely required for the expression of activity, enhancement of Proto accumulation appeared to be less pronounced than was indicated by the high insect mortality. The only total loss of insecticidal activity was observed in 1-(3-sulfopropyl) pyridinium hydroxide (Fig. 18.7, Table 18.14, # 3, 4). QSAR investigations of this compound revealed a 3.3 fold increase in the value of its dipole moment (17.03 D) in comparison to 1,10-diethyl-2,40-cyanine iodide (5.11 D) (Table 18.14, # 7, 8) that lacked a sulfonyl group at the N+ position (Fig 18.7). This was accompanied by a substantial increase in positive charge binding volumes and a substantial decrease in positive charge repelling volumes around the molecule. In other words, it appeared that the presence of an unbalanced positive charge at the N+ position was essential for pyridinium activity. This was also corroborated by the reduced insecticidal activity of Bis-N-methylacridinium nitrate that has an NO3À counterion (Table 18.14, # 5, 6). 18.6.4 Structure-Activity Relationship of Substituted Quinolines and Oxypyridines Structure-activity relationships of four substituted 8-hydroxyquinolines, and two substituted oxypyridines were also investigated (Fig. 18.7). Porphyric insecticidal effects were positively correlated with Proto accumulation, and the presence of both

18.6 Structure-Activity Studies of Porphyric Insecticide Modulators 437 Table 18.14 Effects of ALA, and substituted pyridiniums, 2-oxypyridines and pyrroles on Proto accumulation and larval death in T. ni Entry Template Treatmenta Proto content Larval mortality after (nmol/100 mg 6 days in the greenhouse protein) (%) 1 Pyridinium Control 1.4abcb 1.7a 2 ALA 2.2bcd 3.3a 3 1-(3-Sulfopropyl) 2.4 cd 0.0a pyridinium hydroxide 4 Above + ALA 4.4de 0.0a 5 Bis-N-methylacridinium 5.6e 21.7b nitrate 6 Above + ALA 10.9f 65.0c 7 1,10-Diethyl-2,40-cyanine 58.1 h 75.0c iodide 8 Above + ALA 87.0 h 85.0 cd 9 1,10-Diethyl-4,- 3.5a 96.7d 40-carbocyanine iodide 10 Above + ALA 21.9 g 93.3d 11 Oxypyridine 1,5-Isoquinolinediol 1.0a 1.7a 12 Above + ALA 3.7de 33.0bb 13 Isocarbostyril 1.2ab 1.7a 14 Above + ALA 16.3 fg 86.7 cd 15 Pyrrole 4,5,6,7-Tetrahydroindole 1.3abc 1.7a 16 Above + ALA 2.6 cd 1.7a 17 ter-Butyl 4-acetyl-3,5- 1.3abc 0.0a dimethyl-2-pyrrole- carboxylate 18 Above + ALA 2.7d 20.4b 19 3-Ethyl-2-methyl-4,5,6,7- 0.9a 0.0a tetrahydroindole-4-one 20 Above + ALA 10.0f 83.3 cd Correlation coefficient 0.76 Level of significance 0.1 % Adapted from Gut and Rebeiz Gut et al. (1993) aTreatment consisted of control diet lacking any added ALA or modulator, diet containing 4 mM ALA, 3 mM modulator or 4 mM ALA + 3 mM modulator, in a randomized complete block design with three replicates. Percent larval death was arcsine transformed prior to statistical analysis bMeans followed by the same letter within a column are not significantly different at the 5 % level of significance ALA and modulator were required for activity (Tables 18.14 and 18.15). 8-Hydroxyquinoline and the NO2À substituted analog, 8-hydroxy-5-nitroquinoline in which the hydrogen atom at position 5 of the macrocycle was replaced by an NO2À group was inactive (Fig. 18.7, Table 18.15, # 3–6). When the nitro group was replaced by a sulfonyl group, as in 8-hydroxyquinoline-5-sulfonic acid, substantial insecticidal activity was observed (Fig. 18.7; Table 18.15, # 9, 10). Further substi- tution with a 4-sulfo-1-naphtylazo group at position 7 of the macrocycle as in 8-hydroxy-7-(4-sulfo-1-naphthylazo)-5-quinoline-sulfonic acid resulted in a minor

438 18 Porphyric Insecticides Table 18.15 Effects of ALA, and substituted quinolines on Proto accumulation and larval mortality in T. ni Proto content Larval mortality after 6 days Entry Treatmenta (nmol/100 mg protein) in the greenhouse (%) 1 Control 1.0bb 0.0a 2 ALA 1.7c 0.0a 3 8-Hydroxyquinoline 1.0a 0.0a 4 Above + ALA 1.9c 0.0a 5 8-Hydroxy-5-nitroquinoline 0.7a 0.0a 6 Above + ALA 3.0d 0.0a 7 8-Hydroxy-7-(4-sulfo-1- 1.8c 1.7a naphtylazo)-5-sulfonic acid 8 Above + ALA 41.8 g 76.7b 9 8-Hydroxyquinoline-5- 1.8c 0.0a sulfonic acid 10 Above + ALA 32.1f 81.7b Correlation coefficient 0.85 Level of significance 1% Adapted from Gut et al. (1993) aTreatment consisted of control diet lacking any added ALA or modulator, diet containing 4 mM ALA, 3 mM modulator or 4 mM ALA + 3 mM modulator, in a randomized complete block design with three replicates. Percent larval death was arcsine transformed prior to statistical analysis bMeans followed by the same letter within a column are not significantly different at the 5 % level of significance reduction in activity (Table 18.15, # 7, 8). The presence of a sulfonyl group at position 5 of the macrocycle was an essential requirement for activity. The main effect of this substitution was a significant increase in the electrostatic positive charge binding volumes around the sulfonic acid group. Moving the exocyclic OH group from position 8 to position 1 and moving the endocyclic N atom from position 1 to position 2, as in isocarbostyril, an oxypyridine compound, also resulted in substantial insecticidal activity in compar- ison to 8-hydroxyquinoline (Table 18.14, # 13, 14; Table 18.15 #, 3, 4). This change resulted in an increase in the magnitude of the dipole moment from 2.33 D to 3.53 D, as well as an increase in electron density (from 0.16 to 0.23 kcal/mol) in the unoccupied molecular orbitals in isocarbostyril. Addition of a hydroxyl group at position 5, as in 1,5-isoquinolinediol, diminished considerably the observed insec- ticidal activity (Fig. 18.7; Table 18.14, # 11, 12). This was accompanied by a decrease in dipole moment and further increase in electron density of the unoccu- pied molecular orbitals. 18.6.5 Structure-Activity Relationship of Substituted Pyrroles Structure-activity relationships of three substituted pyrroles were investigated (Fig. 18.7). As was generally observed for the other templates, porphyric insecticidal

References 439 effects were positively correlated with Proto accumulation and the presence of both ALA and modulator were required for activity (Table 18.14, # 19, 20). Highly substituted pyrroles such as tert-butyl 4-acetyl-3,5-dimethyl-2-pyrrole-carboxylate exhibited low insecticidal activity (Fig. 18.7; Table 18.14, # 17, 18). However tetrahydroindoles with an isolated keto group at position 4, as in 3-ethyl-2-methyl- 4,5,6,7-tetrahydroindol-4-one, were active (Fig. 18.7; Table 18.14, # 19, 20). Removal of the ketone group resulted in complete loss of activity as in 4,5,6,7-tetrahydroindole (Fig. 18.7; Table 18.14, # 15, 16). This was accompanied by a substantial decrease in electron density (from 1.03 to 0.52 kcal/mol) of the unoccupied molecular orbitals, and a significant decrease (from 132.3 to 61.8 A3) in volumes of the positive charge binding electrostatic field. 18.7 Epilogue It is hoped that based on the reported investigations it will be possible to develop safe formulation for the control of household pests. It will be more difficult to develop formulations for the control of insects on living plants as the ALA and modulators so far described appear to destroy the plant as well as the insect. References Duggan JX, Gassman M (1974) Induction of porphyrin biosynthesis in etiolated bean leaves by chelators of iron. Plant Physiol 53:206–215 Gut L, Lee K, Juvik JA et al (1993) Porphyric insecticides. IV: structure-activity study of substituted phenanthrolines. Pestic Sci 39:19–30 Gut L, Lee K, Juvik JA et al (1994a) Porphyric insecticides 6. Structure activity study of substituted pyridyls. Pestic Biochem Physiol 50:1–14 Gut LJ, Juvik JL, Rebeiz CA (1994b) Porphyric insecticides. In: Duke SO, Rebeiz CA (eds) Porphyric pesticides, chemistry, toxicology and pharmaceutical applications, vol 559. American Chemical Society, Washington, DC, pp 206–232 Halliwell B (1984) Oxygen-derived species and herbicide action. What’s New in Plant Physiol 15:21–24 Lamola AA, Yamane T (1974) Zinc protoporphyrin in the erythrocytes of patients with lead intoxication and iron deficiency anemia. Science 186:936–938 Lee K, Rebeiz CA (1995) Subcellular localization of protoporphyrin IX and its photodynamic effects on mitochondrial function of the cabbage looper (Trichoplusia ni). In: Heitz JR, Downum KR (eds) Light activated pesticides, vol 616. American Chemical Society, Washington, DC, pp 152–164 Rebeiz CA (1993) Porphyric insecticides. J Photochem Photobiol B Biol 18:97–99 Rebeiz CA (2002) Analysis of intermediates and end products of the chlorophyll biosynthetic pathway. In: Smith A, Witty M (eds) Heme chlorophyll and bilins, methods and protocols. Humana Press, Totowa, pp 111–155 Rebeiz CA, Juvik JA, Rebeiz CC (1988a) Porphyric insecticides 1. Concept and phenomenology. Pestic Biochem Physiol 30:11–27

440 18 Porphyric Insecticides Rebeiz CA, Montazer-Zouhoor A, Mayasich JM et al (1988b) Photodynamic herbicides. Recent developments and molecular basis of selectivity. Crit Rev Plant Sci 6:385–434 Rebeiz CA, Juvik JA, Rebeiz CC et al (1990a) Porphyric insecticides 2. 1,10-Phenanthroline, a potent porphyric insecticide modulator. Pestic Biochem Physiol 36:201–207 Rebeiz CA, Reddy KN, Nandihalli UB et al (1990b) Tetrapyrrole-dependent photodynamic herbicides. Photochem Photobiol 52:1099–1117 Rebeiz CA, Nandihalli UB, Reddy K (1991) Photodynamic herbicides and chlorophyll biosyn- thesis modulators. In: Baker NR, Percival M (eds) Herbicides. Elsevier, Amsterdam, pp 173–208 Rebeiz CA, Gut LJ, K. L et al (1995) Photodynamics of porphyric insecticides. Crit Rev Plant Sci 14:329–366 Waldbauer GA, Cohen RW, Friedman S (1984) An improved procedure for laboratory rearing of the corn earworm heliothus zea (Lepidoptera:Noctuidea). Gt Lakes Entomol 17:113–120

Chapter 19 ALA-Dependent Cancericides I came to realize that life lived to help others is the only one that matters and that it is my duty and my highest and best use as a human. (Adapted from Ben Stein) 19.1 Prologue In 1978, Dougherty and coworkers observed that upon injection of hematoporphyrin derivative (HpD), a chemically synthesized porphyrin molecule, into cancerous tissues the compound accumulated to higher concentrations in malignant tissue than in normal tissues. This finding constituted the basis for use of porphyrins, specifically HpD, in photoradiation cancer therapy. The work was summarized later by Dougherty (Dougherty 1987). Photodynamic Therapy (PDT), has concentrated primarily on the use of hematoporphyrin (Hp) and its derivatives, such as dihydrohematoporphyrin and commercially available Photofrin I and II, which are patented derivatives of Hp. When the photodynamic herbicides technology (see Chap. 17) was described in 1984 (Rebeiz et al. 1984). I received several inquiries from the medical community about the possibility of developing the technology to kill cancer cells. I responded by the affirmative since plants (Rebeiz et al. 1984), insects (Rebeiz et al. 1988) and animals shared the same heme pathway up to protoporphyrin IX (Proto). However due to involvement with photodynamic herbicide and insecticide development I could not immediately work on developing that technology. Then in 1989, Malik et al. investigated the possibility of stimulating endogenous protoporphyrin production by supplementing the medium of transformed cells with ALA (Malik et al. 1989). The K562 cell line did not synthesize Proto during 4 days of incubation with ALA in darkness. However, FELC (Friend erythroleukemia cells) and Bsb (a highly metastic cell line) were sensitive to ALA treatment and synthesized porphyrins in the dark during 4 days of incubation. Trypan blue C.A. Rebeiz, Chlorophyll Biosynthesis and Technological Applications, 441 DOI 10.1007/978-94-007-7134-5_19, © Springer Science+Business Media Dordrecht 2014

442 19 ALA-Dependent Cancericides exclusion and 3H-blue thymidine incorporation were used to determine cell viability and proliferation, respectively. Chromatography was used to determine the type of porphyrin species that accumulated. Flow cytometry was used to determine porphy- rin content by monitoring the red fluorescence (longer than 630 nm) emitted from individual cells. The fluorescence was proportional to porphyrin content. The cells that synthesized Proto became sensitive to light and exhibited 99 % inactivation (cell death) after 4 days of dark incubation at the optimal Proto concentration. Then Kennedy and coworkers (Kennedy et al. 1990; Kennedy and Pottier 1992, 1994) used ALA to treat different types of skin cancers. In their protocol, ALA was mixed into a paste (10 % glaxal base 35–100 mg ALA/lesion), then applied topically to the skin. The skin was then photoirradiated with laser light. The results appeared to be promising. However highly intense light (52 mW/h/cm2) and high concentrations of ALA were required. As a consequence, these conditions caused some damage to healthy tissues. Then it was reported that better ALA-dependent photoradiation therapy results were obtained by using long chain-fatty acid esters of ALA. That in turn reduced the hydrophilic properties of ALA and improved its penetration into treated tissues (Kloek et al. 1996). The ALA esterases that converted the ALA esters into ALA before conversion to Proto were very active in animal tissues and less active in insect and plant tissues (Kolossov and Rebeiz 2004). The rest of this chapter is devoted to the discussion of using ALA with porphyrin modulators for the photodynamic destruction of cancer cells. 19.2 Photodestruction of Tumor Cells by Induction of Protoporphyrin IX Accumulation by ALA and 1,10-Orthophenanthroline Photodynamic herbicides (Chap. 17) and porphyric insecticides (Chap. 18) are two novel technologies that manipulate the photosensitizing capability of metabolic porphyrins. These two novel technologies destroy undesirable plants and insects following co-treatment with δ-aminolevulinic acid (ALA), a naturally occurring 5-carbon amino acid, and one of a number of tetrapyrrole biosynthesis modulators (Rebeiz et al. 1984, 1988). The amino acid and the modulator act in concert. The amino acid serves as a building block for intracellular tetrapyrrole accumulation, while the modulator amplifies the accumulation of harmful tetrapyrroles. In the light, the accumulated tetrapyrroles photosensitize the formation of singlet oxygen which kills treated plants or insects by oxidation of their cellular membranes. In the next four sections it is shown that treatment of rapidly multiplying immortalized cells, with ALA and 1–8-orthophenanthroline (Oph) caused the cells to accumulate much larger amounts of Proto than untreated cells. This endogenous Proto accumulation caused in turn rapid cell death in the light. Slower growing cells responded to such treatments by accumulating much lower levels of Proto.

19.2 Photodestruction of Tumor Cells by Induction of Protoporphyrin IX. . . 443 19.2.1 Identification of the Porphyrin That Accumulated in MLA 144 Cells After Treatment with δÀAminolevulinic Acid and 1,10-Phenanthroline as Protoporphyrin IX Since herbicide and insecticide investigations revealed that Oph induced tetrapyrrole accumulation in plants (Chap. 17) and Proto in insects (Chap. 15) it was conjectured that it may do the same in cancer cells. Therefore Initial investigations attempted to determine whether significant Proto biosynthesis and accumulation could be induced in proliferating MLA 144 gibbon lymphoma cells after incubation in total darkness with 1 mM ALA and 0.75 mM Oph. It was observed that The ALA + Oph treated cells accumulated large amounts of a pigment that exhibited emission and Soret excitation maxima at 298 K and 77 K identical to authentic Proto (Rebeiz et al. 1992). After Mg insertion, the pigment exhibited identical fluorescence properties as Mg-Proto (Rebeiz et al. 1992). To further confirm the identity of the accumulated product, its HPLC mobility was compared to authentic Proto on a Waters Bondapack reverse phase C18-bonded column. Authentic Proto, dissolved in methanol, and the pigment extracted from MLA 144 cells exhibited similar retention times of 5.4–5.6 min (Rebeiz et al. 1992). Since the fluorescence properties of this pigment at 298 K and 77 K were identical to authentic Proto before and after Mg insertion, and since its HPLC mobility was similar to that of authentic Proto, the observed fluorescence was ascribed to the biosynthesis and accumulation of Proto by the treated cells. Protoporphyrin IX accumulation in the treated cells amounted to 29.4 nmol/100 mg of cell protein (Rebeiz et al. 1992). After the same period of dark-incubation Proto was not detected in untreated cells. It was therefore concluded that after treatment with ALA and Oph, MLA 144 cells accumulated large amounts of Proto. 19.2.2 Induction of Cell Lysis of MLA 144 Cells Treated with ALA and Oph Next, the extent of cell destruction in the light following the induction of tetra- pyrrole accumulation by MLA 144 cells was investigated. The cells were labeled with Na51Cr and then induced to accumulate Proto by treatment with 1 mM ALA and 0.75 mM Oph for 3.0 h in darkness. After exposure to white light (sodium halide, 2.11 mW cmÀ2) for an additional 30 min, cell destruction was evaluated by monitoring the release of 51Cr into the incubation medium. ALA + Oph- treated cells that had accumulated Proto exhibited severe photodynamic damage in the light as compared to control cells which had not accumulated any tetrapyrroles (Rebeiz et al. 1992).

444 19 ALA-Dependent Cancericides The relative merits of ALA and Oph in inducing Proto accumulation and cell lysis were also investigated by treatment of MLA 144 cells in darkness either with ALA, with Oph, or with a combination of the two compounds. Although ALA alone induced significant levels of Proto accumulation and photodynamic cell injury, the most dramatic tetrapyrrole accumulation and cell destruction were observed when cells were treated jointly with ALA + Oph (Rebeiz et al. 1992). The addition of Oph doubled Proto accumulation and significantly increased cell lysis in the light. 1,10-Phenanthroline, alone, did not induce Proto accumulation. 19.2.3 Proto-Dependent Photodestruction of MLA and WEHI 164-Clone13 Cells Following ALA and Oph Treatments The susceptibility of other transformed cell lines to ALA + Oph treatment was also investigated. The response of a human granulocytic leukemia cell line (K562), and a murine fibrosarcoma cell line (WEHI 164 clone 13), was compared to that of MLA 144 cells (Rebeiz et al. 1992). Both transformed cell lines exhibited photo- dynamic injury similar to the MLA 144 cells. However, the human granulocytic leukemia cells were extremely sensitive to treatment as evidenced by the lower concentrations of ALA and Oph that induced significant photodynamic cell lysis (Rebeiz et al. 1992). 19.2.4 Enhancement of Proto Accumulation by Murine Splenocyte Treatment Since increased tetrapyrrole biosynthesis and accumulation is more likely to take place in rapidly growing and multiplying cells in need of heme for cytochrome formation, it was conjectured that slowly proliferating cells might be less prone to accumulate tetrapyrroles than rapidly multiplying cells. To test this hypothesis, splenocyte suspensions from BALB/c mice were prepared and, following incuba- tion in the presence or absence of the mitogenic lectin Con A (1.25 pLg/mL for 40 h), were untreated or treated with 1 mM ALA + 0.75 mM Oph for 3.5 h (Rebeiz et al. 1992). The amount of Proto accumulation in these two cell populations was compared to ALA + Oph treated MLA 144 cells. Resting splenocytes treated with ALA + Oph accumulated much less Proto compared to similarly treated Con A-activated splenocytes, which exhibited a 50-fold increase in cell prolife- ration as indicated by tritiated thymidine incorporation. Concanavalin A-activated splenocytes accumulated levels of Proto comparable to those of ALA + Oph treated MLA 144 lymphoma cells.

19.3 Intracellular Localization of Heme Biosynthesis in Animal Cells 445 However resting and Con A-activated splenocytes, as well as MLA 144 cells, treated with medium alone, did not accumulate any detectable amounts of Proto (Rebeiz et al. 1992). 19.3 Intracellular Localization of Heme Biosynthesis in Animal Cells Since Proto accumulation is the essential factor in causing photodynamic damage in animal cells, the next three sections investigated its intracellular accumulation in mammalian cells. The intracellular site of heme biosynthesis in mammalian cells has been investigated by (Granick 1967). Using ox liver mitochondria, Granick proposed the following scheme: ALA is formed in the mitochondria and then translocates out of the mitochondria into the cytoplasm. In the cytoplasm, ALA is converted to copropor- phyrinogen III (Coprogen III). Then Coprogen III translocates back into the mitochon- drion where it is converted to Proto and heme. While this hypothesis has been popular for many years, it is beset with problems. Translocations into and out of the mitochondria are energetically wasteful. Secondly, since no evaluation of mitochondrial breakage during mitochondria isolation has been provided, it was difficult to evaluate contamina- tion of the cytoplasm by mitochondrial content. It was therefore decided to reevaluate this hypothesis in neoplastic cells as described below. 19.3.1 Purity of the Mitochondrial Preparations First the purity of the mitochondrial preparations used in the studies was evaluated. Subcellular organelles were isolated from MLA 144 cells by differential centrifuga- tion in sucrose buffer. To determine the purity of the mitochondrial fraction, the activity of three marker enzymes for mitochondria, cytoplasm and endoplasmic reticulum (ER)/microsomes was determined. Succinate cytochrome c reductase (SCR), a mitochondrial marker located the inner mitochondrial membrane, was used as an indicator of mitochondrial activity. Washed mitochondria exhibited a significant enrichment in SCR activity in comparison to the cell homogenate (Rebeiz et al. 1996a). There was little or no SCR activity in the cytoplasm and ER/microsomes, thus indicating that mitochondria were not present in these fractions (Rebeiz et al. 1996a). In addition, hydroxypyruvate reductase (HPR), an ER/microsomal marker and lactate dehydrogenase, a cytoplasmic marker, were significantly lower in the mitochondrial fraction (Rebeiz et al. 1996a). These results indicated that it was possible to prepare mitochondria from MLA 144 cells with reduced contamination by cytoplasm and ER/microsomal fractions. Next it was determined whether mitochondria were the site of Proto accumula- tion as described below.

446 19 ALA-Dependent Cancericides 19.3.2 Protoporphyrinogen Accumulation in the Mitochondria of MLA 144 Cells Treated with ALA and Oph Homogenates of MLA 144 cells treated with 1 mM ALA and 0.75 mM Oph for 3 h in darkness accumulated significant amounts of protoporphyri(nogen) [Proto(gen)] i.e. a mixture of Proto and its reduced hexahydro analog protoporphyrinogen (Protogen), but did not accumulate other porphyrins. After oxidation of Protogen to Proto, the latter was identified by its fluorescence emission and Soret absorption maxima at 632 nm and 404 nm, respectively, in hexane-extracted acetone at room temperature (Rebeiz et al. 1992). Proto was also identified by chromatography with standard Proto on a Waters Bondapak reverse-phase C18-bonded column (Rebeiz et al. 1992). Following subcellular fractionation of the treated cells, the Proto(gen) content of each subcellular fraction was determined. The total Proto(gen) content of treated cells averaged 221 Æ 18 nmol/100 mg protein (n ¼ 6). The 1,000Âg pellet, which contains mainly nuclei, plasma membranes and unbroken cells accumulated significantly less Proto(gen) (95 Æ 10 nmol/100 mg protein). The bulk of the biosynthesized Proto(gen) accumulated in the mitochondria (235 Æ 45 nmol/100 mg protein, p < 0.05). Some Proto(gen) accumulated in the ER/microsomes (62 Æ 8.7 nmol/100 mg protein), but very little was observed in the cytoplasm (9 Æ 2 nmol/100 mg protein) (Rebeiz et al. 1992). These results confirmed that in MLA 144 cells mitochondria were the primary site of Proto biosynthesis (Dailey 1990). It was conjectured that the Proto found in the ER/microsomes may be due to contamination by broken mitochondria and/or to transport of Proto(gen). 19.3.3 Biosynthetic Origin of Protoporphyrinogen Accumulation in the Mitochondria To determine the biosynthetic origin of Proto(gen) accumulation in the mitochondria, the ability of isolated MLA 144 subcellular fractions to synthesize porphyrin(ogen)s from ALA in the presence of Oph was investigated. In addition to Proto(gen), the formation of other porphyrin(ogen)s was observed. Prior to identification, porphyrinogens were converted to porphyrins as described in (Rebeiz 2002). The oxidized form of coproporphyrinogen (Coprogen), i.e. copropporphyrin (Copro), was identified by its fluorescence emission and Soret excitation at 620 and 394 nm, respectively, in hexane-extracted acetone at room temperature (Rebeiz et al. 1975) and by chromatography with standard Copro on a Waters Bondapak reverse-phase C18-bonded column (Rebeiz et al. 1996a). Other oxidized porphyrinogens such as uroporphyin III (Uro) (RT ¼ 3.4 min) as well as

19.3 Intracellular Localization of Heme Biosynthesis in Animal Cells 447 heptaporphyrins (RT ¼ 4.2 min) and hexaporphyrins (RT ¼ 5.8 min) were formed during the conversion of Urogen to Coprogen. Incubation of MLA 144 whole cell homogenates with ALA and Oph in darkness, resulted in the formation of Proto and lesser amounts of Uro(gen), heptaporphyrin (ogen) and Copro(gen) (Rebeiz et al. 1996a). However, incubation of the cytoplas- mic fraction with ALA + Oph resulted mainly in the biosynthesis and accumulation of Uro(gen), heptaporphyrin(ogen) and Copro(gen), and lesser amounts of Proto (gen) and hexaporphyrin(ogen). On the other hands Isolated mitochondria treated with ALA (1 mM), Oph (0.75 mM) and ATP (15 mM) formed very little Proto(gen) (Rebeiz et al. 1996a). Some Copro(gen) accumulation was observed (10 pmol/5 ml reaction) which might be due to contamination of the mitochondrial fraction by low levels of cytoplasm/ ER. The cytoplasmic/ER fractions accumulated significant amounts of Copro(gen) (25 pmol/5 ml reaction; p < 0.05) and much smaller amounts of Proto(gen). More significantly, when the cytoplasmic/ER and mitochondrial fractions were combined, in addition to Copro(gen), significant amounts of Proto were formed (Rebeiz et al. 1996a). Similar results as those described above were observed when cytoplasm alone, and cytoplasm + mitochondria were incubated with ALA (1 mM), Oph (0.75 mM) and ATP (15 mM), in the absence of added ER. Altogether, these results indicated that Proto biosynthesis and accumulation from ALA in MLA 144 cells required the cooperation of mitochondria and cytoplasm. 19.3.4 Cofactor Requirement for the Biosynthesis and Accumulation of Protogen by Mitochondria To gain better understanding of the cooperation of mitochondria and cytoplasm during the biosynthesis and accumulation of Proto(gen), in mitochondria, the cofactor requirement of this process was investigated. Preliminary experiments indicated that the conversion of ALA to Protogen by mitochondria + cytoplasm was ATP-dependent (Rebeiz et al. 1996a). Thus the possibility that Coprogen, formed in the cytoplasm, may be actively transported into the mitochondria where it is converted to Proto was therefore investigated. Coprogen (1,250 pmol/ ml) was incubated with isolated mitochondria in the presence or absence of ATP. In the absence of ATP, some Proto biosynthesis was observed (29 Æ 11.5 nmol/ 100 mg protein). However, at 100 mM of exogenous ATP, a 62 % increase in Proto formation was observed in comparison to controls (p < 0.05), thus indicating that ATP enhances the conversion of exogenous Coprogen to Proto by mitochondria. The high concentration of added ATP may have been mandated by the high concentration of the added Coprogen substrate.

448 19 ALA-Dependent Cancericides 19.4 Induction of Apoptosis in Leukemia Cells by Modulators of Heme Biosynthesis We have shown in preceding sections that the concurrent use of Oph, a tetrapyrrole heme biosynthesis modulator, with ALA, significantly increases the efficacy of ALA treatment of transformed cells by enhancing Proto accumulation. Heme biosynthesis modulators such as Oph enhance Proto accumulation in transformed cells in the presence of ALA, increase cell lysis in vitro and enhance Proto-induced tumor necrosis in vivo with little damage to surrounding normal tissue (see section below). In insects, modulators have also been associated with the induction of a dark death phenomenon, whereby treated tissues undergo cell death in the absence of light (Chap. 18). The dark cell death, attributed in insects to the formation and accumulation of Zn-Proto has also been observed as well, in cancer cells treated with ALA and Oph. One important possibility in cancer cells is that in addition to enhancing light-induced tumor necrosis by increasing endogenous Proto accumu- lation, Oph might also cause cell death in darkness by inducing programmed cell death, or apoptosis (Fisher 1994). Apoptosis is a type of cell death that exhibits distinct morphological and biochemical characteristics. It plays a key role in the mode of action of a diverse array of anti-tumor agents (Dive and Hickman 1991; Eastman 1990; Lowe et al. 1993; Strasser et al. 1994) and in oncogenesis (Fannidi et al. 1992; Hart et al. 1987; Symonds et al. 1994). In addition, many of the intermediate signaling molecules involved in apoptosis, such as p53, are known to cause growth arrest of neoplastic cells (Radvanyl et al. 1993; Rebeiz et al. 1994; Yonish-Rouach et al. 1991; Zhu and Anasetti 1995), prior to apoptosis induction. One important intracellular mechanism occurring during apoptosis induction is the changes in mitochondrial transmembrane potential caused by the opening of large pores, which are part of the permeability transition (PT) phenomenon (Kroemer et al. 1995). The proteins that form these pores have not been identified, but the mitochondrial peripheral-type benzodiazepine receptor (M-PBR) has been implicated during PT (Kinally et al. 1993; Pastorino et al. 1994). The M-PBR may also be involved in porphyrin/porphyrinogen transport since coproporphyrinogen (Taketani et al. 1994), Proto (McEnery et al. 1992; Verma and Snyder 1988) and heme (Taketani et al. 1995) bind to the M-PBR. We have recently shown that coproporphyrinogen transport into mitochondria is enhanced by ATP (Rebeiz et al. 1996a), and the adenine nucleotide is a component of the M-PBR complex (McEnery et al. 1992). We have therefore investigated the role of apoptosis in the Oph-mediated dark death phenomenon using MLA 144 leukemic T cells. While Oph and Proto, but not ALA, induced growth arrest, only Oph induced apoptosis. The results also suggested that induction of apoptosis by Oph may occur via the M-PBR and that apoptosis contributes to the death of MLA 144 cells in darkness.

19.4 Induction of Apoptosis in Leukemia Cells by Modulators of Heme Biosynthesis 449 19.4.1 Inhibition of DNA Synthesis by Oph Many anti-tumor agents cause cells to pause during the cell cycle and induce growth arrest prior to cell death (Dive and Hickman 1991; Strasser et al. 1994; Yonish-Rouach et al. 1991). We therefore investigated whether ALA, Oph or a combination of the two reagents could reduce cell proliferation. This was done by measuring rates of DNA synthesis. All doses of Oph, but not ALA, caused a significant decrease in DNA synthesis after 3 h of incubation, as evidenced by reduced 3H-thymidine incorporation into the cells. Proliferation of MLA 144 cells was inhibited 64 % at 0.75 mM Oph, a dose that was previously shown to be non-toxic and to enhance Proto accumulation in the presence of ALA in vitro (Rebeiz et al. 1992). The level of inhibition was similar when ALA was used in combination with Oph, at all doses tested. Treatment of MLA 144 cells with ALA alone at any concentration did not significantly alter the rate of 3H-thymidine incorporation. DNA synthesis was also inhibited after 6 h and 24 h incubation with Oph (Rebeiz et al. 2001). 19.4.2 Reduction of Cell Proliferation by Proto and Non-chelating Isomers of Oph Ortel et al. (1993) reported that the metal chelator desferrioxamine inhibited cell proliferation by chelating cellular iron, and this chelation suppressed progression through the cell cycle. To test the possibility that inhibition of cell growth by Oph could be due to its iron-chelating properties, two non-chelating positional-isomers of Oph, 1,7-Oph and 4,7-Oph, were used in a DNA synthesis assay. Exogenous Proto, which has been shown to inhibit the proliferation of cells by binding to (M-PBR) in mitochondria (Verma and Snyder 1988), was used as a positive control. Both Oph isomers inhibited 3H-thymidine incorporation in MLA 144 cells in a dose-dependent manner similar to Oph (Rebeiz et al. 2001). As expected from previous work on exogenous Proto (Verma and Snyder 1988), growth arrest was induced at micromolar concentrations. Surprisingly, exogenous Proto was 10–30 fold more efficient at inhibiting DNA synthesis than Oph or its isomers (p < 0.05). The positional isomer 1,7-Oph was two to four fold more effective than Oph in causing growth arrest of MLA 144 cells (p < 0.05). These results indicated that the iron-chelating property of Oph was not responsible for inducing cell growth arrest. 19.4.3 Cell Viability and Membrane Permeability of MLA 144 Cells Treated with ALA, Oph or Proto Apoptotic cells still have intact plasma membranes, and may appear viable, while necrotic cells quickly lose membrane intactness (Cohen et al. 1992). It was

450 19 ALA-Dependent Cancericides conjectured that the decreased DNA synthesis observed in MLA 144 cells treated with Oph and Proto could be due to a toxic effect of these chemicals. The plasma membrane integrity of MLA 144 cells subjected to trypan blue exclusion (cell viability) and chromium release (membrane permeability) was therefore assessed. Although treatment with Proto and Oph for 3 h in darkness caused growth arrest, cell viability was not significantly reduced as evidenced by trypan blue exclusion. Indeed, the total cell number did not decrease in Oph or Proto treated cells. Proto-treated (0.05–0.1 mM) cell viability after 3 and 24 h of incubation was similar to controls. A chromium release assay was also used as a test of membrane permeability. A combination of ALA and Oph did not induce significant chromium release from cells, during 3 h of incubation with ALA (1–4 mM) and Oph (0.75–3 mM) (Rebeiz et al. 2001). Thus, at 0.75–3 mM, Oph did not appear to be toxic to MLA 144 cells and did not induce cell lysis during 3 h of dark of incubation. However, after 24 h of treatment with Oph (1.5–3 mM), cell membranes no longer excluded trypan blue (15–16 % viability) (Rebeiz et al. 2001). Also, non-chelating isomers of Oph significantly decreased cell viability after 3 and 24 h of incubation. 19.4.4 Induction of Apoptosis by Oph Internucleosomal cleavage of DNA was first measured to confirm the occurrence of apoptosis. MLA 144 cells that had been serum-deprived for 3 h exhibited slight DNA fragmentation in comparison to cells at time zero (Rebeiz et al. 2001). However, treatment with Oph at either 0.75 mM or 1.5 mM induced a dramatic increase in DNA cleavage after 3 h of dark incubation. A similar DNA laddering pattern was observed when cells were treated with a combination of ALA and Oph. Internucleosomal cleavage of DNA was also evident after 6 h incubation with Oph. Treatment with ALA or Proto alone did not induce DNA fragmentation at either 3 or 6 h. Thus, although Proto did induce growth arrest, unlike Oph, it did not induce apoptosis. Cells undergoing apoptosis usually exhibit a characteristic hypodiploid peak of DNA when stained with the DNA intercalating dye PI and analyzed by flow cytometry. Thus MLA 144 cells were treated for 3 h with ALA, Oph, Proto, or medium, in a serum-free system prior to ethanol permeabilization and staining with PI. Cell cycle analysis was determined by analysis with MPLUS software. Basal level of apoptosis was 8 Æ 3.7 % in medium-treated cells (Rebeiz et al. 2001). Treatment with 1.5 mM Oph induced a significant level of apoptosis (45 Æ 1.8 %, p < 0.05), and a distinct subdiploid peak (apoptotic) became apparent. In addition, the proportion of cells accumulating in early S phase increased in comparison to medium-treated cells (60 Æ 2.5 % vs. 48 Æ 2.5 % respectively, p < 0.05). At 0.75 mM Oph, which induced the highest level of internucleosomal DNA cleavage, the majority of the cells became apoptotic (81 Æ 5.6 %), and most of

19.5 Induction of Tumor Necrosis by ALA and Oph-Dependent Photodynamic Therapy 451 the DNA appeared in the hypodiploid peak (Rebeiz et al. 2001). The G0/G1 peak decreased to 9 Æ 0.5 % (p < 0.05) and the G2/M peak became virtually non-existent (0 Æ 0.1 %). A significant majority of the cells appeared to be arrested in early S phase (88 Æ 9.8 %, p < 0.05), a phenomenon often observed in cell cycle dependent apoptosis (Zhu and Anasetti 1995). Neither ALA alone or Proto induced apoptosis, nor did these reagents significantly alter the cell cycle when compared to medium-treated cells (Rebeiz et al. 2001). 19.4.5 Abrogation of Induced Apoptosis by Cycloheximide (Rebeiz et al. 2001) The classical inductive pathway of apoptosis, as observed in thymocytes during negative selection or following treatment with dexamethasone, is defined by the ability of cycloheximide to inhibit its induction (Cohen et al. 1992). To deter- mine whether Oph causes apoptosis via an inductive pathway, MLA 144 cells were incubated for 18 h with cycloheximide (1.5 μg/ml) and then treated for 3 h with varying amounts of Oph in the continuing presence of cycloheximide. Cells were then double-labeled (Cohen et al. 1992) with Hoechst 33342 and PI. In flow cytometric analysis, apoptotic cells are represented by high Hoechst 33342 and low forward angle light scatter (FALS) with the PI positive (necrotic) cells excluded. Oph at concentrations ranging from 0.375 to 1.5 mM induced a significant increase in the proportion of apoptotic cells, with the highest level of apoptosis occurring at 0.75 mM (85 %, p < 0.05). Medium-treated cells, which were serum-deprived for 3 h, were 5 % apoptotic. Cycloheximide alone increased the apoptotic population in medium-treated cells to 15 %, however this was not statistically significant. Co-incubation of cells with cycloheximide and Oph decreased Oph-induced apo- ptosis. The percentage of apoptotic cells was reduced to background levels at all doses of Oph (Rebeiz et al. 2001). These results indicated that Oph causes apoptosis via the classic induction pathway that requires protein synthesis. 19.5 Induction of Tumor Necrosis by ALA and Oph-Dependent Photodynamic Therapy As was mentioned previously the approved use of ALA and its long chain fatty acid esters to treat skin cancer has met with limited acceptance by the medical commu- nity, because of damage to surrounding tissues and limited effectiveness as com- pared to liquid nitrogen treatments (oral communication from my skin doctor). In this section we will describe, as an alternative, the use of AlA and Oph as a model research system for treating Meth-A cancerous cells and solid tumors.

452 19 ALA-Dependent Cancericides The treatment focuses on combined treatment with ALA and Oph in a murine syngeneic solid tumor model using Meth-A cells. It was shown that the combination of ALA and Oph induced the accumulation of porphyrins in Met- A cells and upon exposure to light, significantly reduced the surface area of the tumors and induces their destruction, as determined by their disappearance at both the visual and histological levels. These data supported the possibility that the accumulation of Proto and subsequent photodestruction of tumor cells can be significantly enhanced in vivo by the use of joint AlA an tetrapyrrole modulator 19.5.1 Proto Accumulation in ALA and Oph Treated Meth-A Ascites Cell Suspensions We determined whether treatment of Meth-A cells in vitro with ALA and Oph would result in Proto accumulation. Meth-A ascites cells were treated with 1 mM ALA and 0.75 mM Oph for 3.5 h in darkness. The combination of ALA and Oph induced very significant increases in Proto accumulation (338 nmol/100 mg pro- tein; P < 0.05). Treatment with ALA alone also induced measurable accumulation of Proto (61.73 nmol/100 mg protein) in comparison with control cells, but this was not statistically significant (Rebeiz et al. 1996b). This level of Proto accumulation is approximately six fold lower than the levels that accumulate with ALA + Oph treatment. This finding is also consistent with the specific cell lysis data in which ALA + Oph-induced cell lysis was approximately seven times higher than that arising from treatment with ALA alone (Rebeiz et al. 1996b). It is interesting that the addition of Oph to ALA-treated Meth-A cells induced more Proto accumulation than what was seen previously in MLA 144 cells (Rebeiz et al. 1992). Therefore it was concluded that some cell type specificity was observed because the efficacy of ALA and Oph in stimulating Proto accumulation appeared to vary significantly with cell types. 19.5.2 Sensitivity of Meth-A Cells to ALA and Oph Treatment Since previous results had shown a positive correlation between the amount of Proto accumulation and specific cell lysis upon illumination in several transformed cell lines treated with ALA and Oph (Rebeiz et al. 1992), Meth-A cells were screened for sensitivity to light activated cell lysis after ALA and Oph treatment. There was no apparent cell lysis in darkness (3 h) in Meth-A cells in medium, or in cells treated with I mM ALA or 0.75 mM Oph alone (Rebeiz et al. 1996b). However, there was an increase in cell lysis when cells were treated with both compounds in darkness (P < 0.05) then were exposed to light. Indeed, upon light

19.5 Induction of Tumor Necrosis by ALA and Oph-Dependent Photodynamic Therapy 453 activation (30 min, 3.798 J/cm2), 16.8 % of the 1 mM ALA-treated Meth-A cells were lysed (P < 0.05). When ALA and Oph were used jointly, specific cell lysis increased over seven fold (P < 0.05) compared to the ALA-treated cells. 19.5.3 Proto Accumulation in ALA and Oph Treated Meth-A Solid Tumors In Vivo Preliminary experiments on Proto accumulation in Meth-A tumors in vivo were conducted using a range of ALA (1–10 mM) and Oph (0.75–7.5 mM) doses. In general, the highest concentration yielded Proto accumulation levels >30 nmol/ 100 mg protein whereas 1 mM ALA and 0.75 mM Oph yielded Proto levels of 1.1 nmol/100 mg. Therefore, for the next series of experiments, doses ranging from 2.5 to 10 mM ALA were used at different time points (Rebeiz et al. 1996b). Untreated tumors did not accumulate Proto, whereas significant amounts of Proto accumulated after 3 and 6 h (31 and 17 nmol/100 mg protein, respectively) were observed at the highest dosage of ALA and Oph tested. Twelve hour or more after injection, the levels of accumulated Proto were lower, probably due to degradation by intracellular enzymes (Mattheis and Rebeiz 1977). A similar pattern of decline in Proto accumulation in single-cell suspensions of a T-cell lymphoma (MLA 144) incubated with 0.5 mM ALA and 0.38 mM Oph for 18 h was also observed. The amount of accumulated Proto fell over tenfold at 18 versus 3 h (1.7 Æ 0.16 versus 19.6 Æ 9.90 nmol/100 mg protein; n ¼ 2). Because substantial Proto had accumulated 3 h after injection with ALA and Oph, this time point was used in subsequent experiments. The next series of experiments focused on the enhancement of ALA-induced Proto accumulation by Oph in Meth-A solid tumors (Rebeiz et al. 1996a) Previous studies with Oph had shown that the optimal Oph concentration to use was about 75 % of the used ALA concentration (Rebeiz et al. 1990). As before, medium-treated tumors or tumors treated with Oph alone at 1.8, 3.75, or 7.5 mM did not accumulate any Proto. Oph at two concentrations, 7.50 and 3.75 mM, enhanced Proto accumulation in the pres- ence of 10 (2.7-fold) and 5 (4.7-fold) mM ALA, respectively (P < 0.05). However, at 1.8 mM, Oph did not enhance Proto accumulation when used in conjunction with 1 mM ALA. 19.5.4 Effect of ALA and Oph Treatment on the Size and Histopathology of Meth-A Solid Tumors Having established that Meth-A tumors accumulate Proto after in vivo injection with ALA and Oph the effects of ALA and Oph phototreatment of the tumors were next investigated by histopathological examination of the tumor and the

454 19 ALA-Dependent Cancericides surrounding normal tissue. Treatment groups consisted of mice with tumors treated with medium or ALA and Oph for 3 h in darkness, followed by illumination for 30 min. The initial mean tumor sizes in both control and treated mice (n ¼ 3) were similar. Mice were allocated randomly to treatment groups. Tumors that received ALA and Oph photodynamic therapy (3 h incubation before a 30-min illumination) were significantly reduced 5.4-fold in size compared to control tumors by day 1 and at all subsequent time points (P < 0.05). By days 4 and 5, there were no palpable tumors in 5 of the 6 treated mice. In contrast, the control tumors doubled in size by days 4 and 5 (P < 0.05). On day 1, one of the treated mice sacrificed for histopa- thology appeared to have a palpable tumor, but histopathological examination showed only edema and epidermal necrosis with no evidence of sarcoma tissue present (Rebeiz et al. 1996b). Subsequent histopathology confirmed that in ALA and Oph-phototreated mice, the Meth-A tumor tissue, which is non-metastasizing, was eradicated, except for a small amount of sarcoma tissue in one mouse. In 5 of 6 mice in which the tumor tissue was eradicated, only necrotic sarcoma tissue remained, indicating a high level of tumor necrosis. In addition, one-half of the ALA and Oph-treated mice had panniculitis and epidermal and dermal damage. This is consistent with a local inflammatory response occurring at the site of ALA and Oph injection and is commonly observed in patients treated with ALA photo- dynamic therapy (Grant et al. 1993; Kennedy et al. 1990; Wolf et al. 1993). Neither panniculitis nor tumor necrosis was evident in control mice. Histopathological sections of control tumors and lesions 3 days after ALA and Oph photodynamic therapy were examined. Meth-A tumors were undifferentiated, as reported earlier (Chun and Hoffman 1987). In control mice, there was some necrosis of adjacent dermis related to expansion of the tumor, and the tumor showed muscle invasion, which is typical of established tumors (Rebeiz et al. 1996b). Some panniculitis was apparent, along with necrosis of the epidermis and inflammation of the dermis, sloughing of the skin, and keratinization, which are all indicators of a photodynamic reaction occurring at the site of injection. References Chun M, Hoffman M (1987) Combination immunotherapy of cancer in a mouse model: synergism between tumor necrosis factor and other defense systems. Cancer Res 47:115–118 Cohen JJ, Duke RC, Fadok VAS, Sellins KS (1992) Apoptosis and programmed cell death in immunity. Ann Rev Immunol 10:267–293 Dailey HA (1990) Conversion of coproporphyrinogen to protoheme in higher eukaryotes and bacteria: terminal three enzymes. In: Dailey HA (ed) Biosynthesis of heme and chlorophylls. McGraw-Hill, New York, pp 123–161 Dive C, Hickman JA (1991) Drug-target interactions: only the first step in the commitment to a programmed cell death. Br J Cancer 64:192–196 Dougherty TJ (1987) Photosensitizers: therapy and detection of malignant tumors. Photochem Photobiol 45:879–889

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Index A content, 146, 393–400, 402, 404, 405 Analytical and preparatory techniques, dehydratase, 172, 173 metabolism, 392, 393 65–127 penetration, 394, 396, 401 Abdomens, 424, 425 translocation, 393, 401, 404 Abscissa, 95, 102, 104, 109, 112, 116, 120 Analytical techniques, 23–27, 65–127, 139, Absorbance spectroscopy, 24, 361 Absorption spectrophotometric 185, 217, 344, 361 Angiosperms, 233–235, 312, 314, 317 techniques, 65 Anisotropy, 149, 150 Acceptor, 186–189, 329, 330, 332–334, Antenna Chl-protein complexes, 258, 260, 297, 338, 339, 349, 350 298, 300, 301, 303, 306, 342 Acclimation, 298, 301 Antenna/PS Chl mismatch, 342 Accumulation of Pchlide a, 394, 396, 401 Anthonomus grandis (cotton boll weevil), Acidic (monocarboxylic) biosynthetic 424, 427 route, 269 Anti-tumor agents, 448, 449 Acropetal (upward) translocation, 391 Apoprotein folds, 327, 334, 348 Acrylic, 14, 217, 281 Apoptosis, 448–451 Apoptotic cells, 449, 451 6-hydroxy enantiomers, 218 Applied chromic acid oxidation, 3 hydroxyl, 44 Arabidopsis thaliana, 22, 155, 198, 236, 255 OH, 20, 21, 162, 217, 218, 375, 381, Assembly of pigment-protein complexes, 343 Assembly of thylakoid membranes, 325 418, 438 (A3) (Sx3)/100, 85 Action spectrum, 9, 235 (A4) (Sx4)/100, 88 Active site of cythochrome c oxidase, 419 Attached roots, 390 Active tetrapyrrole metabolism, 396, Authentic concentration ratio, 95, 109, 112 Authentic MV/DV Chl(ide) a ratios, 109 398, 401 [(A3)(X3)/100], 85 Advanced modulator group, 417 [(A4)(X4)/100], 88 Agarose, 160, 161 Axial coordination, 232, 281, 363 Age-dependent, 394, 396, 398, 401 Axially coordinated, 281 Agricultural plan productivity, 337–340 ALA See δ-Aminolevulinic acid (ALA) B Algae, 234, 313 Bacteriochlorophyll, 1, 5, 6, 218, 255, 374 Aluminum foil, 360, 379, 393 BALB/c mice, 444 δ-Aminolevulinic acid (ALA), 10, 27, 57, 58, 65, 127, 131, 146, 167, 168, 170, 186, 189, 311, 329, 330, 362, 394, 395, 400, 442, 443 availability, 393, 396, 398 C.A. Rebeiz, Chlorophyll Biosynthesis and Technological Applications, 457 DOI 10.1007/978-94-007-7134-5, © Springer Science+Business Media Dordrecht 2014

458 Index Barley, 10, 11, 13, 17, 21, 22, 54, 126, 127, C 153, 154, 156, 158, 159, 161, 169, Cabbage looper, 424, 426 178, 185, 187, 190–196, 198, 199, C20 alcohols, 270 201, 202, 204–206, 210–212, 216, Cancer cells, 57, 441–443, 448 220, 222, 225, 227, 228, 230, 234, Cancerous tissue, 441 236, 237, 244, 247, 270, 272, 273, Cancerous tumors, 27 292, 302–304, 313, 318, 321, 331, Carbon skeleton, 337, 338 333, 334, 352, 373 Carboxylic routes, 20, 55, 184, 311 Carolina Biological Supply, 427 BchB, 234, 235 Carotenoids, 2, 39–40, 132, 235, 334, 343, BchG, 255 BchH, 97, 206 348, 351, 364 Bchl, 234, 235 Cell lysis, 128, 443–444, 448, 450, 452, 453 BchM, 206 Cell proliferation, 449 BchN, 206, 234 Cellulose MN, 138 Benzene and phenanthrene, 374 Cell viability, 442, 449–450 1,4-Benzodiazine, 375 Central Mg-atom, 3, 4, 232 Beta-mercaptoethanol, 218 ChemBase™ database, 378 Bi Bi mechanism, 206 Chemical biosynthetic heterogeneity, 53, Biochemical heterogeneity, 289, 326 Bioengineering, 57, 58, 337–354 167, 171, 185, 186, 199, 207, 209, Biosynthetic heterogeneity, 16, 46–48, 53, 55, 219, 221, 223, 225, 240, 325, 345 Chemical derivatization, 48, 50, 52, 198, 199, 167, 170–173, 175, 178, 184–189, 198, 207, 210, 216, 218, 267, 279, 410 199, 206–212, 216, 219–221, 223, 225, Chemical energy, 189, 337, 338 240, 247, 253–262, 289, 291, 295–306, Chemical heterogeneity, 53, 185–186, 311–322, 325–327, 337–354 216–227, 253, 267, 270, 289, 326 Biosynthetic origin, 267, 268, 446–447 Chl See Chlorophyll (Chl) Biosynthetic routes, 16, 19, 20, 48, 50, Chl (E440 F631), 66 53–55, 121, 134, 167, 171, 184–188, Chl(ide) (E440 F640), 67 197–212, 215–230, 233–249, 253–262, Chl(ide) (E440 F676), 67 265–276, 289, 291, 300, 303, 311–313, Chl(ide) a (E672 F680), 249 320, 325, 327, 334, 343, 345, 347, Chl(ide) a (E674 F683), 249 351–354, 363 Chl a acceptors, 189, 330, 332–334, 349, 350 Biota, 338 Chl a-DHGG, 254 Biotronette Mark III Environmental Chl a fluorescence, 364 Chamber, 427 Chl a-GG, 254, 257, 261 Biquinoline, 435 Chl a-hexahydroGG, 254 Blattella germanica, 424, 427, 428 Chlamydomonas reinhardtii, 353, 374 Blue shift, 364, 366–368 Chl a-phytol, 254–257, 260, 261, 301 Boll weevil diet, 427 Chl-apoprotein thylakoid biosynthesis Boll Weevil Research Laboratory, center, 326, 347 USDA, SEA, Mississippi State, Chl-apoprotein thylakoid biosynthesis Mississippi, 427 models, 326, 347 Bovine serum albumin (BSA), 140, 142, Chl a-tetrahydroGG, 254 144, 147, 148, 154, 155, 157–159, ChlB, 235 161, 162 Chl(ide) b(F660 E460], 83 4-Branched Chl a biosynthetic pathway, 18 Chl binding, 326, 327, 346, 351 6-Branched Chl a biosynthetic pathway, 19 Chl biosynthetic routes, 16, 50, 53–60, 184, Broad specificity, 312 185, 188, 237, 300, 313, 320, 327, Broad vibrational emission band, 97 334, 347, 351, 352, 354 Bryophytes, 313 Chl c3, 281 BSA See Bovine serum albumin (BSA ) ChlD, 22 Bx (0–0) Soret excitation, 99 Chl(ide)-free fluorescence amplitudes, 67, 69 Building block, 10, 56, 170, 362, 410, 442 ChlH, 22

Index 459 ChlI, 22 Complexity of photosynthetic membranes, 312 Chlide See Chlorophyllide (Chlide) Con A-activated splenocytes, 444, 445 Chlide a (E668 F674), 248 Concanavalin A-activated splenocytes, 444 Chlide a (E676 F690), 233, 248, 249 Concept and phenomenology, 27, 379, 409 Chlide a (E682 F697), 233, 237, 249 Condition of the glass, 90 Chlide a-geranylgeraniol, 254 Coproporphyrin III (Copro III),, 11, 446 Chlide a H (E668 F674), 247 Coproporphyrinogen III (Coprogen III), 168 ChlL, 235 Corn, 156, 159, 185, 198, 206, 216, 229, Chl-making capabilities, 360 ChlN, 235 235, 244, 261, 262, 270, 272–274, Chlorella, 8, 9, 11, 13, 14, 39, 43, 50, 177, 291, 294, 302, 306, 307, 313, 319, 322, 374, 388, 396, 426 197, 205, 215, 217, 268 Corn earworm, 424 Chlorin e6 trimethyl ester, 7 Correlation, 95, 103, 104, 109, 112, 410–416, Chlorofucine, 6 418, 420, 429, 433–435, 437, 438, 452 Chlorophyllase, 3, 4, 255, 282, 290 Correlation coefficient, 104, 109, 112, 412, Chlorophyll (Chl), 66, 186, 189, 311, 362, 417 413, 415, 434, 435, 437, 438 Chlorophyllide (Chlide), 186 Cotyledons, 35, 36, 39–41, 49, 55, 60, 131, Chloroplast 132, 136–147, 155, 156, 161–163, 198, 202, 206, 208, 222, 229, 234, bioengineering, 57, 58, 337–354 239, 265, 267, 268, 270, 274, 276, membranes, 153–159, 161, 198, 364 291, 292, 297–299, 318, 319, 359, ribosomes, 345 361, 370, 373, 397, 398 Chl-protein biosynthesis, 326, 327, 347 Covalent bond energy, 337 Chl-protein complex 29 (CP29), 178, 189, 327, Cythochrome c oxidase, 418, 419 CP29 See Chl-protein complex 29 (CP29) 330, 332–334, 347, 364, 366–369 CP47 See Chl-protein complex 47 (CP47) Chl-protein complex 47 (CP47), 82, 93, 327, C5-pathway, 183 Cucurbita pepo (pumpkin), 270 330, 333, 334, 347, 364, 366–369 Cyclopentanone ring, 53, 162, 206, 217–219, Chl-protein complex 57 (CP57), xxix 223, 228, 368 Chl-protein complexes, 23, 167, 169, 171, Cytochrome f, 339 Cytochromes, 134, 192, 339, 382, 418, 188, 207, 231, 258, 260, 261, 295, 419, 425, 444, 445 297, 298, 300, 301, 303, 306, 326, Cytometry, 442, 450 331–334, 342, 343, 345–349, Cytoplasm and endoplasmic reticulum 351–354, 363, 364, 367 (ER)/microsomes, 445 Chl synthetase, 255 Cytoplasmic, 176, 345, 445, 447 Chl-thylakoid apoprotein biosynthesis models, 349–351 D Chromatographic mobility, 43, 132, 282, The Dark and light phases, 224, 313 283, 290, 294 Dark death, 379, 382, 411 Chromatophores, 13, 205 Dark-death hypothesis, 418–420 Chromic acid oxidation, 3, 9 Dark Divinyl-Light Divinyl (DDV-LDV), Chromium release, 450 Chromophore-protein, 227–228 198, 313, 318 Cibacron Blue, 160, 161 Dark-Divinyl/Light-Divinyl/ Light-dark Circadian clock, 238 Clark oxygen electrode, 425 Divinyl/Greening group, 318 Cocklebur, 313 Dark incubation, 137, 142, 143, 145, 156, Collision process, 236 Combination of spatial and chemical 223, 241, 254, 257, 261, 297, 305, biosynthetic heterogeneities, 167, 360, 372, 373, 382, 392, 395, 397, 185, 325, 345 399, 405, 410–413, 416–418, 420, Commercialization, 362 421, 425, 427, 433, 442, 443, 450 Common morningglory, 313 Dark Monovinyl (DMV), 198, 313, 318 Common precursor, 134, 135, 188, 269, 330, 349

460 Index Dark Monovinyl-Light Divinyl-Light Dark Dissected, 424 Monovinyl (DMV-LDV-LDMV ), Dithionite, 235 21, 185, 198, 199, 201, 202, 204, 207, Dithiothreitol, 159, 218 210–212, 216, 220–222, 224–227, 229, Divinyl (DV), 186 242–247, 259–261, 270, 291–293, 295, Divinyl Pchlide α (DV Pchlide α), 13, 17, 300–305, 320 19, 46, 48, 49, 54, 152, 154, 155, 159, Dark phases, 219, 221–223, 225, 240, 242, 188, 191, 200, 201, 203, 208–210, 212, 244–246, 257–258, 260–262, 304, 320 216–227, 239–241, 243, 244, 246, 257, 258, 261, 267, 268, 270, 274, 276, 291, Dark phases of the photoperiod, 206, 219, 294, 306, 311–313, 317, 318, 320, 329, 221–223, 225, 240, 244–246, 257, 333, 343, 349, 365, 367–369 258, 260–262 DMV See Dark Monovinyl (DMV) DMV-LDV-LDMV See Dark Monovinyl- Dark tetrapyrrole-dependent death, 390 Light Divinyl-Light Dark Monovinyl DDV-LDV See Dark Divinyl-Light Divinyl (DMV-LDV-LDMV ) DNA laddering, 450 (DDV-LDV) Donor, 15, 169, 186–188, 190–196, 236, DEAE-cellulose, 237 237, 329–331 DEAE-Sephacel, 160, 161 Droplet size, 379 Death, 56, 178, 379–382, 390, 391, 401, Dual pathway, 312 DV Mg-Protos (E424 F591), 92 410–418, 420–423, 425, 427, 434, DVMpe esterification, 267 435, 437, 438, 448, 449 DV Pchlide α See Divinyl Pchlide α Decline in cereal yield, 337 (DV Pchlide α) Decline of PORA, 237 DV tetrapyrrole, 90, 312, 343, 371, 372 Decrease in oxygen consumption, 425 Dynamic phenomena, 59 Degenerate, 187, 329 Demonstration of metabolic pathways, 279 E Dermal damage, 130, 454 Early intermediate, 330, 365 Descriptors and biological activity, 429 Edema and epidermal necrosis, 454 Desferrioxamine, 449 EDTA, 140, 142, 144, 145, 147, 148, 151–153, Destructive free radicals, 419 Dexamethasone, 451 155, 157, 159–162 DHGG See Dihydrogeranylgeraniol (DHGG) E417 F587, 93, 94, 96 1,2-Diazine, 375 E424 F591, 92–94, 96 Dicarboxylic and monocarboxylic E440 F631, 66 tetrapyrroles, 311, 418 E440 F670, 66 1,1’-Diethyl-4,4’-carbocyanine iodide, 436 E’440 F’640, 83 The Diet of T. ni, 420 E’440 F’676, 67, 68 Dihydrogeranylgeraniol (DHGG), Electron and proton flow, 339 17, 254, 297 Electron density, 434, 438, 439 Dihydrohematoporphyrind, 441 Electron transport, 3, 338, 342, 418 3-Dimensional chemical structures, 429 Electron transport chains, 342 4,4’-Dimethyl-Dpy, 435 Electrostatic Diphenyl ether herbicides, 27, 178 Dipole-dipole interaction, 186, 188, 329 binding, 433 2,2,-Dipyridyl, 375, 410 interaction, 429 2,3-Dipyridyl, 375 potential energy levels, 429 2,4-Dipyridyl, 375 volumes, 429 4,4’-Dipyridyl, 375 Emission (F) amplitudes, 91 4,4’-diphenyl-Dpy, 435 Endogenous, 20, 134, 144–146, 157, 163, α,α-dipyridyl (Dpy), 13, 169, 190, 198, 199, 205, 206, 208, 410, 412–417, 419–421, 255, 372, 441, 442, 448 424, 425, 429, 434–436 Endoplasmic reticulum (ER), 247–249, Dipyridyl ketoxime, 435 Direct esterification, 20, 258, 260 445–447 Disruption of the LHCI-730, 368

Index 461 Energy transfer, 23, 57, 167, 169, 171, 178, Fluorescence excitation (E), 91, 107 186–190, 199, 209, 220, 223, 225, 227, Fluorescence polarization, 149, 150 236, 240, 249, 327, 329–334, 346, 347, Fluorescence spectroscopy, 15, 44, 66–67, 349–351, 364, 366 71, 197, 199, 202, 206, 210, 216, Enhancers of ALA conversion, 372 218, 223, 267, 268, 361, 363 Environmental complications, 337 Fluorescence techniques, 45, 65, 304, 374 Epidermal damage, 130, 454 Fluorescence was proportional to ER See Endoplasmic reticulum (ER) porphyrin content, 442 Esterification, 3, 20, 53, 142, 162, 205, Folding, 326, 327, 334, 346, 348, 351 Food, 337–339, 354, 420–422, 424, 253–258, 260–262, 267, 268, 272, 426, 427 297, 361, 365, 367 Fractional photoconversion, 247 Esterification process, 253 Free radical damage, 419 Ethyl, 8, 48, 53–54, 201, 202, 210, 211, French bean, 313, 321 215, 217, 223–226, 241, 244, 245, Frozen in liquid nitrogen, 396 268, 311, 312, 369 Full sunlight, 341, 342 Etio, 281 Fully esterified Chl a biosynthetic route, 265 Etiolated, 10, 39, 90, 131, 185, 216, 237, Fully esterified route, 269 253, 265, 279, 290, 311, 344, 359 Fully greened, 238 Etioplast, 136, 141, 145, 153, 157–160, 162, 254 Eubacterial nitrogenase, 235 G Euglena gracilis, 253, 267 G4, 255 Eukaryotic cells, 418 GA See Gibberellic acid (GA) Euphotic zone, 262, 283, 284, 294, 306, 312 Gabaculine, 171 Evolutionary ancestor, 234, 313 Gas-chromatographic /mass spectroscopic Evolutionary intermediate, 313 Excited donor “D*”, 186, 187, 329 analysis, 267 Exogenous tetrapyrroles, 359, 365–370 General purpose simultaneous equations, Experimental error, 122, 212, 288 Experimental MV/DV ratio, 109 105, 114, 118 Extensive desiccation, 411 Geranylgeraniol (GG), 17, 163, 249, 254, Extrinsic factors, 338, 341, 354 262, 270 F Geranylgeraniol diphosphate (GGDP), 163 Farnesol, 267, 270 German cockroach, 424, 426 Farnesyl-PP, 255 GG See Geranylgeraniol (GG) Fat bodies, 424, 425 GG-PP, 255 FeCl2, 422 Gibberellic acid (GA), 142, 143, 162, 163 FeCl3, 422 Glasses of diethyl ether, 92 Female guts, 424, 425 Glutamate semialdehyde (GSA), 171, 172 Ferns, 234, 313 Glutamate t-RNA Ligase, 172 Ferrochelatase, 12, 183, 419 Glutamate t-RNA (Oxido) Reductases, 172 Ferrous iron, 12, 183, 419 Glutamic acid, 15 Fiber, 337, 354 Glutamyl-tRNA complex, 171 Field conditions, 239, 339–342, 370, 393, Grana membranes, 366 Greenhouse-grown, 379, 393–401, 406 394, 406 Greening Field desorption mass spectroscopy, 216 Field-grown plants, 393–400, 406 group affiliation, 20, 56, 208, 234, Fifth-instar T. ni larvae, 425, 426 313–317, 320, 322, 371 Fischer, H., 4–6, 48, 167, 268 Flow cytometry, 442 groups, 22, 60, 185, 313–317, 320, Fluorescence amplitude, 66, 67, 69–71, 92–96, 344, 370–373, 379 106–109, 111–112, 114, 116, 120, 149 Growth arrest, 448–450 GSA See Glutamate semialdehyde (GSA) GSA-Aminotransferase, 172 Gymnosperms, 233, 234, 313

462 Index H 8-Hydroxyquinoline, 13, 46, 374, 375, 380, Harderopoprphyrinogen, 176 428, 429, 436–438 HAT See Hydroxyaminotetrahydropyranone Hydroxy pyruvate reductase (HDR), xxix (HAT) 8-Hydroxyquinoline-5-sulfonic acid, 437, 438 3H-blue thymidine incorporation, 442 Hydroxy radical, 418 Heliothus zea, 424–428 Hypocotyl hooks, 141, 143, 144, 147, 161 Hematoporphyrin derivative (HPD), 27, 441 Hypocotyls, 35, 141, 143, 144, 147, 161, 391 HemC, 173 Hypodiploid peak, 450, 451 HemD, 174 Hypogenous, 391 Heme, 2, 7, 39, 57, 167, 170, 172, 173, I 178, 183, 188, 189, 196, 330, 349, Imidazole N, 366 362, 379, 382, 410, 417, 419, 425, Incident solar radiation, 340 433, 441, 444–451 Inducer-enhancer of Proto accumulation, 418 Heme metabolic pathway, 362, 410 Inducers of tetrapyrrole accumulation, 372 Hemolymph and gut, 423 Infra-red, 7 Heptaporphyrins, 175, 447 Ingestion, 420 Herbicide industry, 56, 359 Inhibitors of MV Pchlide accumulation, 372 Heterogeneities, 53, 167, 185, 198, 325, 345 Insect death, 418, 419, 427 Heterogeneity of thylakoid membranes, Insects, 57, 362, 379, 382, 390, 392, 410, 325–329 Hexacoordinated, 92, 99, 363, 366, 369 412, 415, 417–420, 422–428, 436, Hexahydro analog 439, 441–443, 448 emission, 446 Instars, 410–412, 414–417, 419–428 soret, 446 Integuments, 423, 424 Hexane-extracted acetone fraction, 67, 68, Interconnected pathways, 287, 291 70, 143, 145, 149, 157 Internucleosomal cleavage, 450 Hexaporphyrins, 175, 447 Intracellular release, 419 Hexahydro Geranylgeraniol (HHGG), xxix Intra-membrane environments, 189, 330, 350 High energy bonds, 338 Intrinsic, 338, 341–342 Higher efficiency of the PETS, 339 Isocarbostyril, 428, 437, 438 Highly intense, 442 Isozymes, 199, 200, 207, 209, 219, 221, 223, High-pressure liquid chromatographic 225, 236, 240 analysis, 267 Hill reaction, 342 J Histidine nitrogen of apoproteins, 232 Jimsonweed, 313, 394 Histidine residues, 366 Johnsongrass, 270, 313, 318, 373, 379, 14-h light/10-h dark regime, 409 HMBL See Hydroxymethylbilane (HMBL) 383–390 Homogenate, 39, 131–138, 144, 147, 155, 157, 162, 360, 445 K HPD See Hematoporphyrin derivative (HPD) Keto derivatives, 14, 217 HPLC analysis, 272, 273 Keto propionate, 217 HPR See Hydroxypyruvate reductase (HPR) Kinetic analysis, 14, 121–131, 287, 291 Human control, 341 77º K MV/DV fluorescence ratios, 105 Human granulocytic leukemia cell line, 444 Hydrogen donor, 15, 236, 237 L Hydroxyaminotetrahydropyranone (HAT), Lactate dehydrogenase, 445 75, 171 Lambsquarter, 313 1-Hydroxymethylbilane (HMBL), 16, 174 Larval extract, 410 [7-Hydroxymethyl]-chlorophyll b, 21, Layer of biosynthetic heterogeneity, 253 289, 290 8-Hydroxy-5-nitroquinoline, 386, 437, 438 Hydroxypyruvate reductase (HPR), 445

Index 463 LCFA See Long chain fatty alcohol (LCFA) M LDDV See Light–dark Divinyl (LDDV) Macrocycle, 3–6, 8, 14, 48, 53–55, 135, 178, LDV See Light Divinyl (LDV) LHCI See Light harvesting Chl-protein 183, 189, 197, 198, 201–203, 205, 207, 210, 211, 215, 217, 218, 223–229, 233, complex I (LHCI) 244, 245, 255, 258, 262, 265, 267, 268, LHCI-680 See Light harvesting Chl-protein 270, 272, 280, 281, 289, 311, 330, 349, 367–369, 396, 433–438 complex 680 of PSI (LHCI-680) Maize shoots, 254 LHCI-730 See Light harvesting Chl-protein Malignant tissue, 441 Malpighian tubules, 424 complex 730 of PSI (LHCI-730) Marker enzymes for mitochondria, 445 LHCII See Light harvesting Chl-protein Maximal quantum efficiency, 340, 341 Mean per cent error (X1), 80, 81, 83, 85, 88, 89 complex II (LHCII) Medical community, 441, 451 LHC See Light harvesting Chl-protein 6-Membered N-heterocyclic compounds, 426 5-Membered N-heterocyclics, 379, 383, 426 complexes (LHC) Membrane-bound, 36, 140, 141, 147, 150–152, Light cycles, 219, 220, 229, 239, 241, 244 154, 156, 158, 197, 246, 338, 345, Light–dark Divinyl (LDDV), 185, 198, 216, 367–369 Membrane intactness, 449 228, 229, 318, 320 Membranes fractions, 148, 150, 197 Light–dark Monovinyl (LDMV), 321 Metabolic activity, 361, 393 Light death, 382 Metabolic fate of ALA, 394 Light-dependent, 229, 236–239, 247, Meth-A ascites cells, 127, 452 Methine bridge, 9 248, 255 Methionine, 134, 142, 145, 148, 152, 154, 162 Light Divinyl (LDV), 198, 313, 318 Mg-containing tetrapyrroles, 362 Light environments, 298, 301 Mg-porphyrins, 15, 25, 45, 50, 72–75, 151, Light harvesting Chl-protein complexes 177, 183, 197 Mg-Proto diester (Mpde), 265–276 (LHC), 178, 189, 299, 326, 327, 330, Mg-Proto ester and/or diester (Mpd(e)), 332, 334, 345, 347, 351, 364, 365 154, 265–275 Light harvesting Chl-protein complex I Mg-Proto monoester (Mpe), 13–16, 18, 54, (LHCI), 178, 189, 327, 330, 332–334, 55, 74, 91, 94–96, 104, 132–135, 347, 364–369 139–142, 148, 153, 162, 177, 186, Light harvesting Chl-protein complex II 197, 205–206, 210–212, 218, 231, (LHCII), 178, 189, 258, 260, 298, 300, 244, 303, 311, 374, 397 301, 303, 327, 330, 332–334, 347, Mg-Protos, 90, 92, 94, 95, 97, 101, 267 350–354, 364–369 Midguts, 424, 425 Light harvesting Chl-protein complex Mitochondria, 13, 136, 170, 176, 178, 183, 680 of PSI (LHCI-680), 178, 189, 330, 396, 418, 425, 426, 445–449 333, 334, 364, 366–369 Mitochondrial damage, 426 Light harvesting Chl-protein complex Mitochondrial function, 426 730 of PSI (LHCI-730), 189, 327, Mitochondrial marker, 426 330–334, 347, 364–369 Mitochondrial peripheral-type benzodiazepine Light-independent, 233, 234, 272, 274 receptor (M-PBR), 448 Light-independent Chlide a E biosynthetic Mitogenic lectin Con A, 444 step, 274 Mixed DV-MV routes, 311 Light-induced protease, 236 Mixtures of MV and DV Pchl(ides), 91 Light phases of the photoperiod, 216, 219, 222, Mixtures of MV DV MPE, 91, 92 224, 225, 239, 243, 244, 247, 258–260, MNDO or PM1 Hamiltonians, 429 297–305, 313 Model for young field-grown plants, 393 Lipids, 38, 40, 41, 343 Long chain fatty alcohol (LCFA ), 253, 258, 262, 270 Long wavelength (LW), 142, 228, 229, 233, 235, 237–242, 244, 247–249, 256–258, 260–262, 330, 350, 369 Loss of body fluids, 411

464 Index Mode of action, 14, 372–374, 409, 417, Necrotic sarcoma tissue, 454 423, 448 Negative potential energy contour lines, 429 Negative potential energy levels, 429 Modulators, 56, 57, 362, 365, 370–400, Negative selection, 451 406, 410, 411, 417–424, 426–439, Neoplastic cells, 445, 448 442, 448–452 Net fluorescence signals, 91, 97, 105, 106, Molar extinction coefficients, 26 114, 118, 119 Monocar-boxylic phorbin, 282 N-ethylmaleimide, 218 Mono-oxygenase, 289, 290 Net photosynthetic efficiency, 340 Monovinyl (MV), 8, 46, 90, 152, 184, 197, New cuticle, 416 Nicotinamide, 140, 339, 374, 375, 379, 380 215, 268, 279, 289, 313, 329, 343, 362 Nicotinic, 374, 375, 379–381 Monovinyl Pchlide (MV Pchlide), 9, 46, 97, Nitrogenase, 234, 235 NMR See Nuclear magnetic resonance (NMR) 152, 188, 198, 216, 241, 256, 268, n-Octyl-B-D-glucoside, 237 289, 311, 330, 343, 369 Non-fluorescing Chl-polypeptides, 364 Mosses, 234 Non-homogeneous glasses, 90 M-PBR See Mitochondrial peripheral-type Nontransformable Pchlide a (nt-Pchlide a), xxx benzodiazepine receptor (M-PBR) Nontransformable short wavelength Mpd(e)) See Mg-Proto ester and/or diester (Mpd(e)) Pchlide a (nt-SW Pchlide a), 234, 237 Mpde esterases, 267, 268 nt-Pchl(ide) a (E628 F630), 229 Mpe See Mg-Proto monoester (MPE) nt-SW Pchl (ide) a H (E440 F630), 229 MPLUS software, 450 Nuclear DNA, 345 Multibranched, 20, 22, 55–56, 185, 279, Nuclear magnetic resonance (NMR), 7, 19, 326–329, 332–334, 343–345, 347 Multibranched biosynthetic pathway 20, 48, 50, 52, 216, 218, 223, 279, (MBP)-sublocation model, 326 281, 284 Multienzyme system, 135 Nucleophyllic amino acid side chains, 232 Multiple regression, 429 Multiple vinyl-reductases, 312 O Multiplicity, 312 10-OH-Chl a lactone, 20 Multistep process, 279, 289 OH radicals, 418 Murine fibrosarcoma cell line, 444 Old cuticle, 416 Murine syngeneic solid tumor, 452 Older greenhouse-grown plants, 393 Mustard, 313 Oncogenesis, 448 MV See Monovinyl (MV) One quadrant, 376 MV and DV Mg-Protos pair, 92 One-window technique, 65 MV Mg-Protos (E417 F589), 92 Open frames, 234 MV Mpe esterification, 268 Ordinate, 95, 103, 104, 109, 110, 112, 116, MV Pchlide See Monovinyl Pchlide (MV Pchlide) 120, 122 MV tetrapyrrole, 90, 184, 312, 343, 371 Organelle, 311 Organello, 16, 55, 65–67, 72, 75, 131–136, N NADH-cytochrome, 426 139–145, 147, 154, 155, 163, 177, 185, NADH dehydrogenase, 426 196, 198, 199, 202, 206, 218, 226, 240, NADPH, 15, 16, 21, 54, 134, 152, 154–158, 246, 344, 365, 368 Organic life, 196, 338 171, 192, 202, 218, 229, 233, 237–238, Organized structure, 367–369 246, 249, 255, 338, 339 1,10-Orthophenanthroline (Oph), 127, 394, NADPH:Glu-tRNA(oxido)reductase, 171 417–426, 429, 433–436, 442–454 Natural metabolic intermediate, 419 Osmotic shock, 149 Nec 2, 18, 52–53, 282–284, 293, 294, 306, 307 Oxorhodo, 281 Nec 2 maize mutant, 282, 283, 293 2-Oxypyridine, 375, 380, 428, 437 Necrotic cells, 449, 451 4-Oxopyridine, 375

Index 465 P Photodynamic herbicides, 27, 56–58, 359–406, Palpable tumors, 130, 454 409, 417, 426, 441, 442 Panniculitis damage, 130, 131, 454 Partially-reduced intermediates, 418 Photodynamic insecticides, 27, 57 Partial overlap, 79 Photodynamic kill, 379 Pattern of Proto accumulation, 410 Photodynamic therapy (PDT), 27, 57, Pattern of tetrapyrrole accumulation, 57, 362, 441, 451–454 372, 406, 410 Photo electron transport system (PETS), PBG deaminase, 173 Pchl H See Protochlorophyll holochrome 338–342, 356 (Found as Photosynthetic electron transport system) (Pchl H) Photoenzyme, 14, 233, 237, 312 Pchl-holochrome, 10, 12, 231 Photointermediate, 249 Pchlide a cyclases, 221 Photoirradiated, 442 Pchlide a-Hochrome (E650 F657) Photoperiods, 216, 219–225, 229, 236, 239–241, 243–247, 256–262, 271, (t-LW-Pchlide a-H (E650 F657), 229 272, 280, 297–305, 313, 318, 320, Pchlide and/or Pchlide ester (Pchl(ide)), 360, 394, 411–413, 416, 420, 422, 427 Photoperiodic greening, 222, 226, 229, 41, 134 239, 241, 244, 271, 303 Pchlide a phytyl ester, 8–10, 13, 14, 19, 268 Photoprecursor, 254, 268, 271 Pchlide-apoprotein complex, 227 Photoradiation cancer therapy, 441 Pchlide cyclase(s), 218, 225 Photoradiation therapy (PDT), 442 Pchlide E (E440 F631), 66 Photoreduction, 226, 233, 237–239, 241, Pchlide ester (Pchlide E), 49, 55, 66, 67 244, 247–249, 312 Pchlide Oxidoreductase A (PORA), 22, 222, Photosensitizing, 56, 359, 442 capability, 442 236–239, 241, 244, 247, 249, 256, herbicidal technology, 359 257, 260 Photosynthesis, 5, 58, 59, 189, 192, 193, Protochlorophyllide (Pchlide), 186 203, 310, 337–339, 341–342, 345, Pentacoordinated, 92, 93, 99, 101, 267, 367 353, 360, 364 Percoll gradients, 425 Photosynthetic efficiency, 23, 337–342 Percoll-purified, 148, 149, 152, 153, 159 Photosynthetic electron transport system Percoll-purified mitochondria, 425 (PETS), 338–342, 354 Permeability transition (PT), 448 Photosynthetic particles, 353 Phenanthrene, 374, 433, 434 Photosynthetic unit (PSU), 326–329, 332–334, Phenanthridine, 374 337, 340–352, 354 1,7-Phenanthroline, 374 Photosystem I (PSI), 178, 189, 236, 257, 258, 1,10-Phenanthroline, 374, 375, 380, 390, 260, 261, 297, 299, 305, 306, 316, 326, 394, 417–423, 428, 434, 442–445 327, 330, 332, 333, 338–342, 346, 347, 4,7-Phenanthroline, 375 350–354, 364 Phenanthrolines, 374, 375, 430, 433–434 Phytol, 3, 4, 20, 75, 135, 145, 163, 249, Phenomenology, 27, 362, 379, 409, 422, 423 253–258, 260, 261, 267, 270, 290, Pheo(phorbide) a (F674 E4121], 81 297, 301 Pheoporphyrin, 281 Phytol-PP, 255 Phorbin, 136, 138, 167, 168, 276, 282, 290 Picolinic, 374–376, 381 Photoconversion, 11, 12, 230, 233, 235–236, Picoplankton, 262, 283, 284, 294, 306, 239–249, 272, 274–276, 292, 318 307, 312 Photoconverted, 232, 233, 237, 244, 246, Pigment-pigment interaction, 232 256, 257, 261 Pigment-protein environment, 363 Photodestruction, 342, 366–369, 392, 412, Plant productivity, 337, 339 442–445, 452 Plant roots, 382, 390 Photodynamic cancericides, 58 Plasma membranes, 446, 449, 450 Photodynamic control of insects, 409 Plastid DNA, 345 Photodynamic damage to mitochondria., 425 Plastid isolation, 147–148, 155, 156 Photodynamic destruction, 58, 362, 442 Photodynamic herbicidal phenomenon, 178

466 Index Plastocyanin, 339 Protoheme, 4, 9, 10, 183, 184, 382, 454 Plastoquinone electron acceptor pools, 338 Protopheophytin, 132, 133, 280–282 Pogostemon cablin, 313, 316 Protopheophytin a adduct, 282 Poisoned porphyrin-heme metabolism, 419 Proto photosensitization, 366 Polarographically, 382, 425 Protoporphyrin, 3, 50, 71, 132, 183, 197, 311, Polypeptide, 206, 237, 334, 348, 351, 363, 329, 343, 361, 409, 441 364, 366 Protoporphyrin IX oxygenase (Protox), Poor ALA penetration, 393 Population increase, 337 15, 177, 178, 265, 365 PORA See Pchlide Oxidoreductase A (PORA) Protoporphyrinogen, 446–447 PorA, 22 Protoporphyrinogen IX oxidase, 15, 177 PorB, 22, 226, 236, 238, 239, 244, 258, 260 Protoporphyrinogen IX (Protogen) oxidase, PORB monomer, 238 PorC, 236–239 15, 27, 168, 175–178, 365, 446, 447 Porphin, 167, 168, 281 Protoporphyrin IX (Proto), 186 Porphyric insecticides, 57, 58, 409–439, Pseudoionone, 4 PSI See Photosystem I (PSI) 441, 442 Photosystem II (PSII), 347 Porphyrin ester synthetases, 269 PSU See Photosynthetic unit (PSU) Porphyrinogens, 11, 446 PT See Permeability transition (PT) Porphyrins, 1–9, 11, 15, 24, 25, 32, 43, Pumpkin seed coat, 5, 6, 48 2-Pyridyl aldehyde and picolinic acid, 374 50, 72–75, 132, 138, 139, 151, 177, 2-Pyridyl aldoxime, 374 178, 183, 189, 197, 363, 441, 442, Pyrrole, 2, 4, 9, 90, 177, 183, 281, 375–379, 446, 452 Position 7 of the macrocycle, 3, 4, 53, 189, 218, 386, 387, 391, 428, 429, 439, 443 227, 258, 262, 267, 330, 349, 437 Positive charge binding, 429, 433, 436, 439 Q Positive potential energy contour lines, 429 QSAR See Quantitative structure activity Positive potential energy levels, 429 Post-spray dark incubation period, 392, 412, relationship (QSAR) 416, 417 Quanta, 235 Pre-accumulation, 392 Quantitative structure activity relationship Precursor-product relationships, 20, 26, 39, 41, 43–46, 55, 121, 122, 126, 222, 267–269, (QSAR), 429, 436 271, 279, 287–289, 291–294, 297, 299, Quantum efficiency of ONE, 340, 341 301, 302, 307 Quantum process, 235 Premature release of O2, 418 Quantum yield, 95, 235–236 Preuroporphyrinogen, 16, 174 Prickly sida, 313 R Primary photochemical act, 338–339 Random, 90, 175, 208, 210, 326, 347 Primitive modulator group, 417 Rank of the instar, 415 Primitive plant species, 313 Rate of photoconversion, 236 Prolamellar bodie(s), 236, 239, 255 Ratio of MV to DV fluorescence signals, 90 Proliferation, 442, 449 Reaction center (RC), 333, 338, 339, 341, Prolonged contact, 406 Pronounced fluorescence signals, 105, 114 342, 350, 366 Prosthetic group Reconstituted systems, 235 catalases, 382 Red fluorescence, 229, 249, 442 peroxidases, 382 Redox-carriers, 342 Prothylakoid membranes, 140, 141, 255 Red-root pigweed, 313 Protochlorophyll (Pchl), 8, 66 Reduction kinetics, 238 Protochlorophyll holochrome (Pchl H), 32 Relative merits, 444 Protogen See Protoporphyrinogen IX Repelling, 429, 433, 434, 436 (Protogen) Repelling electrostatic volumes, 429 Resting splenocytes, 444 Restricted collision, 236

Index 467 Rhodapseudomonasspheroides, 13, 15, 46, 48, Specific cell lysis, 452, 453 170, 177, 197, 205, 206, 218, 374 Specific insecticidal applications, 409 Specific radioactivity, 122–125, 127, 135, Rhodobacter capsulatus, 202, 258, 312 Rhodofying, 281 136, 138, 139, 287, 288, 291 Rhodopseudomonas spheroids, 46, 170, Spectral shifts, 233, 237, 247–249 Spectrofluorometric, 15, 24–26, 66, 67, 72–75, 177, 205 Ribosomes, 345 105, 113–114, 118–121, 139, 157, Ring substitution, 435 161, 265, 270, 272, 273, 280, 282, Rotational movement, 236 290, 361, 410 Route, 12, 48, 121, 134, 167, 184, 197, 215, Spectrofluorometric analysis, 105, 113–121, 139, 272, 273, 280, 361 233, 253, 265, 289, 311, 325, 343, 373 Spinach, 11, 178, 255, 301 Spin restrictions, 418 S Splenocyte suspensions, 444 S-Adenosyl methionine (SAM), 205, 206 Split Soret excitation, 106 S-Adenosyl methionine methyl transferase Splitting of Soret excitation bands, 106 Spraying, 410, 417, 420 (SAMMAT), 205–210 Stage of greening, 145, 368, 369 Sarcoma tissue, 454 State of aggregation, 368, 369 Scenedesmus obliquus, 170 Static phenomena, 59 SCR See Succinate cytochrome c Stoichiometric, 258 Stroma fractions, 148–153, 155–157, 161, reductase (SCR) 171, 193, 194, 333, 350 Seed coat of Cucurbitaceae, 270 Stromal factor, 218 Semi-aqueous environment, 367 Structural complementarity, 433 Shibata shift, 12 Structural similarities, 270, 271, 275 Short wavelength (SW), 20, 66, 71, 92, Structure-function analyses, 429 Structure-function studies, 426, 429 100, 138, 228, 330 Subcenters, 326, 327, 334, 346, 348, 349, 351 Shuttling, 14, 40, 233, 237, 334, 351 Subdiploid peak, 450 Shuttling photoenzyme, 233, 237 Substituted pyridiniums, 432, 436, 437 Signal deconvolution, 106 Substituted pyrroles, 376, 438–439 Significant larval mortality, 411 Succinate cytochrome c reductase (SCR), 445 Silica Gel H, 132, 133, 135, 137, 138 Succinate oxidase, 426 Single-branched pathway (SBP)-multilocation Succinyl-thiokinase, 170 4-Sulfo-1-naphtylazo group, 437 model, 326–328, 334, 346–348, 351 Sulfonyl group, 436–438 Single-branched pathway (SBP)-single Superoxide radical, 418 SW See Short wavelength (SW) location model, 326, 327, 332, 346, (Sx1), 79–81 347, 350 (Sx1)/100, 79 Singlet oxygen, 57, 362, 382, 406, 410, Sx2, 83, 84, 86, 88 418, 419, 442 Synechocystis, 193, 282, 353 Singlet tetrapyrroles, 57 Synergistic effects, 412–415 Site of tetrapyrrole accumulation, 423 Skin cancers, 442 T Slow metabolism, 393 T-cell lymphoma, 453 Smaller PSUs, 343, 345, 352–354 TDPH modulators, 371–379, 390–400 Small leaf section, 396 Temperature-dependence, 158 Solar energy, 189, 337, 338 Template of Chl-protein biosynthesis centers, Soret excitation maximum, 66, 71, 76, 92, 93, 99, 106, 114, 366–370 326, 327, 334, 347, 351 Southern andNorthern analyses, 255 Ternary complex, 229, 233 Soybean, 198, 313, 370, 371, 373, 394 Spatial biosynthetic heterogeneity, 53, 167, 185, 199, 207, 209, 219, 221, 223, 225, 240, 253, 325, 345 Spatial heterogeneity, 171, 178

468 Index 2,2’:6’2\"-terpyridine, 435 t-SW Pchl(ide) a-H (E443 F640), 229 Tetraahydrogeranylgeraniol (THGG), 17, 254, t-SW Pchl(ide) a-H (E444 F636), 229 t-SW Pchl(ide) a-H (E445 F640), 229 270, 297 Tumor necrosis, 448, 451–454 Tetrapyrrole-dependent photodynamic Tumor sizes, 454 Turnover rate, 342 herbicides (TDPH), 57, 361, 362, Two dimensional and three dimensional 370–400, 406, 409 Tetrapyrroles computer modeling, 373 accumulation, 57, 361, 362, 370, 372, 375, Two photon process, 247 379, 390–406, 410, 418, 420–423, 433, Two windows technique, 65 442, 444 donors, 188, 189, 329, 330, 332, 349, 350 U THGG See Tetraahydrogeranylgeraniol Ultracentrifugation, 148, 150, 151 (THGG) Unexcited acceptor “A”, 186, 187, 329 Thin-layer chromatographic, 24 Unfolded state, 326, 345, 346, 351 Thioflavin T, 401–404, 428 Uro See Uroporphyrin (Uro) Third instar T. ni., 410 Urogen See Uroporphyrinogen (Urogen) Three-banded emission chloroplast profile, 367 Urogen decarboxylase, 175 Thylakoid Urogen III synthase, 173 assembly, 25, 325 Uroporphyrin (Uro), 11, 24, 45, 446, 447 biosynthesis sub-centers, 326 Uroporphyrinogen (Urogen), 12, 168, environments, 167, 171, 199, 207, 217, 219, 220, 223, 225 172–175, 447 Thymocytes, 451 Tightly bound, 150, 178, 419 V t-LW Pchl(ide) a (E447 F657), 229 4VChlR See 4-Vinyl Chl a reductase (4VChlR) t-LW Pchl(ide) a (E450 F657), 229 4VCR See 4-Vinyl Chlide a reductase (4VCR) Topography of photosynthetic membranes, 23 Velvetleaf, 313, 394 [(t-Pchl(ide) a E639 F00)], 229 Vibrational, 97, 236 [t-(Pchl (ide) a E650 F655)], 229, 247 Vinyl, 8, 21, 48, 53–54, 176, 197, 198, 201, t-Pchlide a. SeeTransformable Pchlide a (t–Pchlide a) 202, 207, 210, 211, 215, 216, 218, Tracheal branches, 424 223–226, 244, 245, 267, 268, 311, 369 Transcribed, 236 4-Vinyl Chl a reductase (4VChlR), 22, 55, Transformable (t), 233 186, 202 Transformable long wavelength Pchlide 4-Vinyl Chlide a reductase (4VCR), 19, 21, a holochrome (t-LW Pchlide a H), 26, 153, 154, 156, 158–161, 186, 187, 233, 237–239, 247, 249 202, 245, 247, 257, 258 Transformable Pchlide a (t-Pchlide a), 235, 247 4-Vinyl Mg-Proto monoester reductase Transformable short wavelength Pchlide (4VMPER), 54, 154, 186, 187, 225, a holochrome (t-SW Pchlide a H), 227, 242, 244, 260, 303, 304 233, 237–239, 247, 249 4-Vinyl Mg-Proto reductase (4VMPR), Trans-hydrogens, 233, 272 22, 153, 186, 202, 210, 211, 224 Transition dipoles, 187 4-Vinyl Pchlide a reductase (4VPideR), Translated, 236, 345 158, 186, 202, 223, 225, 226, 244, 312 Translocation barrier, 406 Vinyl pheoporphyrin, 6 Trichoplusia ni (T. ni), 410 4-Vinyl reductase (4VR), 152, 153, 161, Triplet state, 57 186, 312, 327, 334, 348, 351 Tropical and temperate oceans, 262, 283, Violent convulsions, 420 284, 294, 312 Viscosity, 236 Trypan blue, 441, 450 Vomiting, 420 t-SW Pchl(ide) a-H (E441 F633), 229 4VMPER See 4-Vinyl Mg-Proto monoester t-SW Pchl(ide) a-H (E442 F636), 229 reductase (4VMPER) t-SW Pchl(ide) a-H (E443 F633), 229

Index 469 4VMPR See 4-Vinyl Mg-Proto reductase X (4VMPR) X(Ea Fb), 91 Xiaojian Dend, 60 4VPideR See 4-Vinyl Pchlide a reductase Xylem, 391 (4VPideR) 4VR See 4-Vinyl reductase (4VR) W Y Waldbauer’s medium, 426, 427, 433 Y(Ec Fd), 91, 93, 97–99, 101, 106, 107, Waters Bondapack reverse phase C18-bonded 111, 115, 119 column, 443 Wheat, 12, 39, 154, 185, 197, Z ZnCl2, 422 216, 244, 254, 270, 292, Zn-porphyrins, 25 313, 321, 322 Zn-Proto, 16, 17, 72–75, 95, 152, 192, Wild oat, 313 World population, 337, 338 418–420, 422, 448