Microsomal metabolism of dibenz[a,c]anthracene, dibenz[a,h]anthracene and dibenz[a,j]anthracene to bis-dihydrodiols and polyhydroxylated products

Polar, ethyl acetate soluble metabolites formed in incubations of dibenz[a,c]-anthracene (DB[a,c]A), dibenz[a,h]anthracene (DB[a,h]A) and the related DB[a,h]A 3,4-diol and dibenz[a,j]anthracene (DB[a,j]A) with 3-methylcholanthrene (3-MC)-induced rat liver microsomal preparations have been separated by HPLC and examined using fluorescence, UV and NMR spectroscopy. Metabolites with spectral properties consistant with their identification as the 3,4:8,9-bis-diol of DB[a,j]A and a 1,2,3,4,12,13-hexol derived from DB[a,c]A were found. DB[a,h]A was metabolized to three polar products identified as the 3,4:10,11-bis-diol and the related 1,2,3,4,8,9- and 1,2,3,4,10,11-hexols, which were also formed, together with the related 1,2,3,4-tetrol, from the DB[a,h]A 3,4-diol. The possible role of bis-diols in the metabolic activation of these three dibenzanthracenes is discussed.     

Exciton interaction among chlorophyll molecules in bacteriochlorophyll a proteins and bacteriochlorophyll a reaction center complexes from green bacteria

Absorption and CD spectra of bacteriochlorophyll a proteins and bacteriochlorophyll a reaction center complexes from two strains of Chlorobium limicola were recorded at 77 °K. Visual inspection showed that the Q_y-band of chlorophyll in either protein was split into at least five components. Analysis of the spectra in terms of asymmetric Gaussian component pairs by means of computer program GAMET showed that six components are necessary to fit the spectra from strain 2K. These six components are ascribed to an exciton interaction between the seven bacteriochlorophyll a molecules in each subunit. The clear difference between the exciton splitting in the two bacteriochlorophyll a proteins shows that the arrangement of the chlorophyll molecules in each subunit must be slightly different.

The spectra for the bacteriochlorophyll a reaction center complexes have a component at 834 nm (absorption) and 832 nm (CD) which does not appear in the spectra of the bacteriochlorophyll a proteins. The new component is ascribed to a reaction center complex which is combined with bacteriochlorophyll a proteins to form the bacteriochlorophyll a reaction center complex. The complete absorption (or CD) spectrum for a given bacteriochlorophyll a reaction center complex can be described to a first approximation in terms of the absorption (or CD) spectrum for the corresponding bacteriochlorophyll a protein plus the new component ascribed to the reaction center complex.     

Absorbance and fluorescence properties of the bacteriochlorophyll a reaction center complex and bacteriochlorophyll a protein in green bacteria

Absorbance, emission and excitation spectra were measured at both room and liquid-nitrogen temperatures for a photochemically active bacteriochlorophyll a reaction center complex and a bacteriochlorophyll a protein isolated from Chlorobium limicola and Chlorobium thiosulfatophilum. The low-temperature absorbance spectrum for the complex has a band centered at 833 nm, which is not seen in the spectrum of the bacteriochlorophyll a protein. We attribute this difference to a modification of the bacteriochlorophyll a protein in the active complex. The room-temperature fluorescence spectra for the bacteriochlorophyll a protein and the complex are similar, as are those measured at low temperatures. The 833-nm component of the low-temperature absorbance spectrum of the complex is relatively nonfluorescent.     

The oxidation of chlorophyll a in alcohols

Oxidation potentials for the chlorophyll (Chl/Chl⁺) couple, relative to that of the Fe²⁺/Fe³⁺ couple, were determined in methanol, ethanol, and isopropanol from the initial extent of reaction of chlorophyll with FeCl₃. The presence of 4,7-dimethylphenanthroline was necessary to get sufficient oxidation of chlorophyll in isopropanol. The values were +0.75 V, +0.865 V, and approx. +1.0 V, respectively, based on an assumed value of +0.77 V for the Fe²⁺/Fe³⁺ potential. It is suggested that alcohols, especially methanol, may stabilize Chl⁺ by adding to the carbonyl group or the conjugated double bond system.     

Coordinate expression of light-harvesting chlorophyll a/b gene family of photosystem II and chlorophyll a oxygenase gene regulated by salt-induced phosphorylation in Dunaliella salina

As a stress factor, salt induces the phosphorylation of light-harvesting chlorophyll (Chl) a/b proteins (LHCII) in Dunaliella salina. In this study, we found that the salt-induced phosphorylation of LHCII was not affected by phosphatase, and that salt simultaneously regulated both the phosphorylation of LHCII and the expression of genes encoding light-harvesting Chl a/b proteins of photosystem II (lhcb) and the gene encoding Chl a oxygenase (cao) in dark-adapted D. salina. The mRNA accumulation patterns of lhcb and cao were similar, which further affected the size of LHCII and the ratio of Chl a to Chl b. Therefore, we inferred this simultaneous regulation is one of the mechanisms of D. salina to adapt to the high-salinity environment.     

The induction of sister-chromatid exchanges in Chinese hamster ovary cells by K-region epoxides and some dihydrodiols derived from benz[a]anthracene, dibenz[a,c]anthracene and dibenz[a,h]anthracene

Experiments were performed to investigate the effects of 3 polycyclic aromatic hydrocarbons, benz[a]anthracene, dibenz[a,c]anthracene and dibenz[a,h]anthracene and K-regio epoxides and some of their related dihydrodiols on the chromosomes of Chinese hamster ovary cells in vitro. Of the 3 hydrocarbons only benz[a]anthracene showed any activity in inducing sister-chromatid exchanges. The K-region epoxide and the 3,4-dihydrodiol have been found to be more active than the corresponding K-region or the other non K-region dihydrodiols derived from benz[a]anthracene. Athough dibenz[a,c]anthracene was almost inactive, the K-region 5,6-epoxide and all 3 possible dihydrodiols, the 1,2-, 3,4- and 10,11-diols were active in inducing increased numbers of sister-chromatid exchanges in the chromosomes of these cells. The 3,4-dihydrodiol of dibenz[a,h]anthrecene was also active in inducing sister-chromatid exchanges whereas the 1,2- and 5,6-dihydrodiols were only weakly active. This study provides some support for the suggestiion that the activation of these 3 hydrocarbons proceeds by the metabolic conversion of non K-region dihydrodiols into vicinal diol-epoxides.     

Dap ( D efective a leurone p igmentation) mutations affect maize aleurone development

Dap (Defective aleurone pigmentation) is the designation for mutations in maize that give rise to a characteristic dappled endosperm phenotype, consisting of patches of purple tissue, of variable size and shape, on a yellow background. Features shared by all Dap mutants are: dominant expression when they are maternally derived, lack of expression or transmission when they originate from pollen, failure to recover homozygous Dap genotypes, reduced frequency of Dap seeds in the progeny of outcrosses of Dap/+ females, association of the dappled phenotype with reduction in seed size. The mutants so far tested, six in all, can be grouped into two classes, one including male-transmissible (MT) isolates not expressed in the endosperm if their contribution is paternal, and a second class of isolates (NMT) that are permanently lost following paternal transmission. We suggest that the NMT mutations are on a chromosome that carries an intercalary deletion. Assuming linkage between the mutant and the deletion, selection against the deficient chromosome during male gametogenesis would account for the failure to recover Dap seeds in the progeny of Dap/+ male parents. We have obtained genetic evidence supporting this hypothesis. This interpretation, however, does not apply to MT alleles. For these, other mechanisms, such as imprinting and/or dosage effects may be proposed. The mutable pattern in the endosperm to which all Dap mutants give rise is an intriguing phenotype which remains to be clarified. An unexpected finding is that aleuronic and subaleuronic cells corresponding to the colourless areas are abnormal in shape and anthocyanin biosynthesis is blocked in these cells. This finding calls for further investigation in light of a possible connection between flavonoid precursors and cell shape. Received: 2 January 1997 / Accepted: 26 May 1997     

pH control of the chlorophyll a fluorescence in algae

The pH of the suspension medium was found to have a remarkable influence on the “slow” (min) time course of Chlorophyll a fluorescence yield in the green alga Chlorella pyrenoidosa and in the blue-green alga Anacystis nidulans. In Chlorella, the decay of fluorescence yield, in the 1- to 5-min region, is strongly retarded at alkaline pH; this decay rate shows an optimum at pH 6–7. In Anacystis, the rise of fluorescence yield, in the same time range, is decreased optimally at pH 6–7; poisoning with 3(3,4-dichlorophenyl)-1,1-dimethylurea reverses the direction of this pH effect. These observations suggest a correlation of the H⁺ status (or the processes associated with it such as photophosphorylation and resulting conformational changes) of the chloroplast to the yield of chlorophyll a fluorescence in vivo.     

Arrangement of the light-harvesting chlorophyll a/b protein complex in the thylakoid membrane

The transverse orientation of the light-harvesting chlorophyll a/b protein complex of Photosystem II (LHC II) in the thylakoid membrane of pea was investigated using surface radioiodination with Iodo-Gen^TM. The labelling effects on LHC II of four different membrane preparations were compared. One preparation was oriented right-side-out (intact thylakoids); two of them had an inside-out orientation exposing the lumenal surface (inside-out vesicles; PS II particles) and one had both sides of the membrane exposed (mechanically damaged thylakoids). It was found that LHC II could be iodinated only in membrane preparations with an exposed lumenal surface. Isolated apoproteins were chemically cleaved. Fragments analysis revealed a tyrosine residue located eight amino acids from the C-terminus as the single iodination site. It is concluded that the C-terminus of LHC II points towards the lumental side of the thylakoid. Differences in the labelling behaviour of the LHC apoproteins could be assigned to a heterogeneity in the C-terminal region in which the tyrosine residue is replaced by phenylalanine.     

The distance between cytochromes a and a3 in the azide compound of bovine-heart cytochrome oxidase

The electron-spin relaxation rates of the two species of cytochrome a³⁺₃-azide found in the azide compound of bovine-heart cytochrome oxidase were measured by progressive microwave saturation at T = 10 K. It has been shown previously that Cyt a⁺³₃-azide gives rise to two distinct EPR resonances, depending upon the oxidation state of Cyt a. When Cyt a is ferrous, Cyt a³⁺₃-azide has g = 2.88, 2.19 and 1.64; upon oxidation of Cyt a, the a³⁺₃-azide g-values become g = 2.77, 2.18, and 1.74 (Goodman, G. (1984) J. Biol. Chem. 259, 15094–15099). The relaxation effect of Cyt a on Cyt a₃ could be measured as the difference in microwave field saturation parameter H_1/2 between the g = 2.77 and g = 2.88 species. For each signal the spin-lattice relaxation time T₁ was determined from H_1/2 using the transverse relaxation time T₂. The value of T₂ at 10 K was extrapolated from a plot of line-width vs. temperature at higher temperature. The dipolar contribution to T₁ was related to the Cyt a-Cyt a₃ spin-spin distance utilizing available information on the relative orientation of Cyt a₃-azide and Cyt a (Erecinska, M., Wilson, D.F. and Blasie, J.K. (1979) Biochim. Biophys. Acta 545, 352–364). By taking into account the relaxation parameters for both g_x and g_z components of the Cyt a₃-azide g-tensor, the angle between the g_z components of the Cyt a and Cyt a₃g-tensors was determined to be between 0 and 18°, and the Cyt a-Cyt a₃ spin-spin distance was found to be 19 ± 8 Å.     

Chlorophyll a fluorescence and photochemical activities of chloroplast fragments

1. Comparative studies were made on the fluorescence characteristics of chlorophyll a at 20° and −193°, and quantum efficiencies for P 700 oxidation and NADP⁺ reduction were measured in chloroplasts and chloroplast fragments obtained after incubation with 0.5% digitonin.

2. Differences in the flurescence yield of chlorophyll a in flowing and stationary suspensions of untreated chloroplasts and of the large fragments are indicative of light-induced photoreduction of the quencher Q of chlorophyll a, associated with pigment System 2 (chlorophyll a₂). The relatively low constant fluorescence yield of chlorophyll a in the small fragments indicates the absence of fluorescent chlorophyll a₂ from these fragments and suggests that the low fluorescence is due to chlorophyll a, associated with pigmen System 1 (chlorophyll a₁). The ratio of the fluorescence yields of chlorophyll a₁ and chlorophyll a₂ is 0.45:1. In the large particles the concentration ratio of pigment System 1 and System 2 is 1:3.

3. The efficiencies of quanta absorbed at 673, 683 and 705 nm for NADP⁺ reduction and P 700 oxidation in untreated chloroplasts and chloroplast fragments indicate that digitonin treatment results in a separation of System 2 from System 1 in the small fragments. Sonication does not cause such a separation. Under the conditions used P 700 oxidation and NADP⁺ reduction in the small fragments separated after digitonin treatment, occurred with maximal efficiency of 0.7 to 1.0 and 0.7, respectively.

4. The constancy of the fluorescence yield of chlorophyll a₁ in the small fragments, under conditions at which P 700 is oxidized and NADP⁺ is reduced, is interpreted as evidence either for the hypothesis that the fluorescence of chlorophyll a₁ is controlled by the redox state of the primary photoreductant XH, or alternatively for the hypothesis that energy transfer from fluorescent chlorophyll a₁ to P 700 goes via an intrinsically weak fluorescent, still unknown, chlorophyll-like pigment.

5. The low-temperature emission band around 730 nm is argued not to be due to excitation by System 1 only; the relatively large half width of the band, as compared to the emission bands at 683 and 696 nm, suggests that it is possibly due to overlapping emission bands of different pigments.     

Unusual patterns of benzo[a]pyrene metabolites and DNA-benzo[a]pyrene adducts produced by human placental microsomes in vitro

Human placental microsomes were incubated with [³H]benzo[a]pyrene (BP) and Salmon sperm DNA and the resulting metabolite-nucleoside complexes resolved by Sephadex LH-20 chromatography. The metabolite pattern was analyzed by high-pressure liquid chromatography (HPLC). The incubates were also co-chromatographed with extracts obtained from incubates with rat liver microsomes and [¹⁴C]BP. Phenols, quinones and 7,8-dihydrodiol were detected in the placental incubates. Both 9,10- and 4,5-dihydrodiols were very low as compared with control rat liver samples. Placental microsomes catalyzed the binding of BP metabolites to DNA in vitro, giving rise to two main complexes which co-chromatographed with rat liver-produced peaks attributable to 7,8-diol-9,10-epoxide and 7,8-oxide and/or quinones when metabolized further. The nucleoside metabolite peaks attributable to 4,5-oxide and 9-phenol-4,5-oxide were lacking when compared with the binding pattern catalyzed by rat liver. Both the total binding and specific metabolite-nucleoside adducts in the placenta correlated with fluorometrically measured aryl hydrocarbon hydroxylase (AHH) activity and with the amount of dihydrodiol formed. The results demonstrate that both the metabolite pattern and the nucleoside-metabolite complexes formed by the placental microsomes in vitro differed greatly from those produced by rat liver microsomes. These studies also suggest that it is not possible to predict specific patterns of DNA binding from AHH measurements or even from BP metabolite patterns, especially when comparing different tissues and species.     

Evidence for the role of the light-harvesting chlorophyll a/b protein complex in photosystem II heterogeneity

Analyses of chlorophyll fluorescence induction kinetics from DCMU-poisoned thylakoids were used to examine the contribution of the light-harvesting chlorophyll a/b protein complex (LHCP) to Photosystem II (PS II) heterogeneity. Thylakoids excited with 450 nm radiation exhibited fluorescence induction kinetics characteristic of major contributions from both PS II and PS II_β centres. On excitation at 550 nm the major contribution was from PS II_β centres, that from PS II centres was only minimal. Mg²⁺ depletion had negligible effect on the induction kinetics of thylakoids excited with 550 nm radiation, however, as expected, with 450 nm excitation a loss of the PS II component was observed. Thylakoids from a chlorophyll-b-less barley mutant exhibited similar induction kinetics with 450 and 550 nm excitation, which were characteristic of PS II_β centres being the major contributors; the PS II contribution was minimal. The fluorescence induction kinetics of wheat thylakoids at two different developmental stages, which exhibited different amounts of thylakoid appression but similar chlorophyll a/b ratios and thus similar PS II:LHCP ratios, showed no appreciable differences in the relative contributions of PS II and PS II_β centres. Mg²⁺ depletion had similar effects on the two thylakoid preparations. These data lead to the conclusion that it is the PS II:LHCP ratio, and probably not thylakoid appression, that is the major determinant of the relative contributions of PS II and PS II_β to the fluorescence induction kinetics. PS II characteristics are produced by LHCP association with PS II, whereas PS II_β characteristic can be generated by either disconnecting LHCP from PS II or by preferentially exciting PS II relative to LHCP.     

Induction of sister-chromatid exchanges in Chinese hamster ovary cells treated in vitro with non-k-region dihydrodiols of 7-methylbenz[a]anthracene and benzo[a]pyrene

Studies were carried out on the incidence of sister-chromatid exchanges induced in Chinese hamster ovary cells by in vitro treatment with the polycyclic aromatic hydrocarbons 7-methylbenz[a]anthracene and benzo[a]pyrene and with related K-region and non-K-region dihydrodiols. Appreciable increases in the incidence of sister-chromatid exchanges were apparent in cells treated with non-K-region dihydrodiols: the most active compounds were 3,4-dihydro-3,4-dihydroxy-7-methylbenz[a]anthracene and 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene and the effects were dose-dependent. The parent hydrocarbons and the related K-region dihydrodiols induced some sister-chromatid exchanges but they were considerably less active than these two non-K-region diols. The results suggest that this system may usefully be applied to studies aimed at determining which dihydrodiols are important in the metabolic activation of the carcinogenic polycyclic hydrocarbons. These and other results also infer that Chinese hamster ovary cells possess some intrinsic ability to metabolize such compounds in the absence of exogenous activation systems.     

Imaging chlorophyll a fluorescence reveals specific spatial distributions under different stress conditions

Chlorophyll a fluorescence has been adopted as a fast, non-invasive, and cheap method to detect stress effects in plants. The majority of these chl-fluorescence measurements have been carried out with ‘clamping’ fluorometers recording punctual chlorophyll a fluorescence at isolated parts of the leaf. However, this method is inherently limited in providing information on the homogeneity of responses to stresses at the leaf or whole plant level. Therefore the purpose of this study was to measure imaging chlorophyll a fluorescence and to compare the temporal and spatial distribution of this emission under allelochemical (2-3H-benzoxazolinone and 3,4-dihydroxybenzaldehyde), thermal and salt, and heavy metal (cadmium, copper and zinc) treatment in the model plant Arabidopsis thaliana (L.) Heynh. The results suggested different spatial distributions for each condition: the two allelochemicals showed inhibition spots at the edges of the oldest leaves and both did not affect the photosynthetic activity of young leaves; treatment with the three heavy metals revealed highly homogenous effects over the whole plant with a quite uniform decrease of maximum PSII efficiency (also in youngest leaves). On the contrary, temperature (heat and cold) and salt stress showed an initial decrease of fluorescence in the tissues around the vascular bundles that lasted between 2 and 3 h depending on the treatment. These irregularities in chlorophyll fluorescence make it difficult to correlate punctual measures (typical for clamping fluorometers) with the effect on the whole plant, ignoring effects that are evident when imaging is used. Therefore these results show that monitoring chlorophyll a fluorescence by imaging improves the measurement of stress effects on treated plants, suggesting that punctual fluorescence measurements do not always reveal the heterogeneity of the stress-related effects in treated plants.     

Calcium-induced formation of chlorophyll b and light-harvesting chlorophyll a/b-protein complex in cucumber cotyledons in the dark

Dark-grown cucumber seedlings were exposed to intermittent light (2 min light and 98 min dark) and then cotyledons were incubated with 50 mM CaCl₂ in the dark. Chlorophyll (Chl) a was selectively accumulated under intermittent light and Chl b was accumulated during the subsequent dark incubation with CaCl₂. The change in chlorophyll-protein complexes during Chl b accumulation induced by CaCl₂ in the dark was investigated by SDS-polyacrylamide gel electrophoresis. Chlorophyll-protein complex I and free chlorophyll were major chlorophyll-containing bands of the cotyledons intermittently illuminated 10 times. When these cotyledons were incubated with CaCl₂ in the dark, the light-harvesting Chl a/b-protein complex was formed. When the number of intermittent illumination periods was extended to 55, small amounts of Chl b and light-harvesting Chl a/b-protein complex were recognized at the end of intermittent light treatment, and these two pigments were further increased during the subsequent incubation of the cotyledons with CaCl₂ in the dark compared to water controls.     

Characteristics of microsomal reduction of benzo[a]pyrene 4,5-oxide

NADPH-reduction of benzo[a]pyrene 4,5-oxide (BP-4,5-oxide) to BP required four components from rat liver: cytochrome P-450, NADPH cytochrome P-450 reductase, phosphatidylcholine and a soluble, heat-sensitive factor which was present in 105 000 × g supernatant and was also released from microsomes by sonication. The requirement for this factor contrasts with recently reported results from Sugiura et al. (Cancer Res., 40 (1980) 2910). Oxide-reduction was 40 times faster under anaerobic conditions, but oxygen did not affect the stimulation factor. This stimulation was highest (× 15) at low concentrations of microsomal protein (<0.1 mg/ml) and was almost absent at high concentrations of microsomal protein (>1 mg/ml). Oxide-reduction activity was proportional to microsomal protein concentration in the presence of added 105 000 × g supernatant, but for microsomes alone (>0.1 mg/ml) exhibited a parallel plot with an intercept at 0.08 mg/ml microsomal protein. Stimulation was highest at high concentrations of BP-4,5-oxide and a linear plot of V⁻¹ vs. [BP-4,5-oxide]⁻¹ was only obtained in the presence of 105 000 × g supernatant (K_m = 3 μM, V_max = 3.3 nmol/mg/min). Microsomal hydration of BP-4,5-oxide (inhibited in reductase assays) was unaffected by 105 000 × g supernatant, suggesting that stimulation of oxide-reduction did not derive from solubilization of BP-4,5-oxide. Stimulation was observed in the initial rate of reaction and was independent of incubation time. Inhibition of lipid peroxidation, removal of peroxides and deoxygenation were all excluded as explanations of the stimulatory effect.     

Temperature dependence of optical properties of chlorophyll a incorporated into phosphatidylcholine liposomes

Temperature-dependent spectral changes of chlorophyll a (Chl a) incorporated into liposomes of two types of phosphatidylcholine are studied. When Chl a incorporated into the liposomes is cooled down to 5°C from the temperature of the gel-to-liquid crystalline phase transition of the lipid, the red shift as well as the increase in half-bandwidth of the red peak of Chl a are only slight. By measuring the difference spectra produced by substracting the absorption spectrum at the phase transition temperature of the lipid from that at lower temperature, it is shown that the component absorbing at longer wavelength (675–685 nm) than the peak of the red maximum (about 670 nm) significantly increases at the expense of the component absorbing at shorter wavelength (657–668 nm). The positions of positive and negative peaks depend on the temperature and the molar ratio of the lipid to Chl a. The absorbance change is most pronounced on cooling below the phase transition temperature of the lipid. The temperature-induced absorbance change is almost completely reversible. The results indicate that the aggregated forms of Chl a in liposomes can be spectrophotometrically detected in the gel phase of the lipid.