Induction of benzo[a]pyrene Mono-oxygenase in liver cell culture by the photochemical generation of active oxygen species. Evidence for the involvement of singlet oxygen and the formation of a stable inducing intermediate.

1. The photochemical generation of excited states of oxygen in liver cell culture by the mild ilumination of culture medium containing riboflavin, results in stimulation of benzo[a]pyrene 3-mono-oxygenase, a cytochrome P-450-linked mono-oxygenase. 2. The same large increase in mono-oxygenase activity was found when medium containing riboflavin was illuminated in the absence of cells and then stored in the dark for 24h before contact with the cells. From this it may be inferred that stimulation is due to the formation of a stable inducer in the culture medium. Further experiments indicate that the stable inducer is due to the photo-oxidation of an amino acid. 3. Evidence that singlet oxygen is responsible for initiating the stimulation of the mono-oxygenase is based on the use of molecules that scavenge particular active oxygen species. Of all the scavengers tested, only those that scavenge single oxygen inhibited the stimulation. 4. A hypothesis is developed to relate the stimulation of the mono-oxygenase by singlet oxygen in cultured cells to the regulation of the cytochrome P-450 enzyme system in vivo. It is suggested that single oxygen generation within cells may be a common factor linking the many structurally diverse inducers of the enzyme system.     

Comparison of the photochemical and enzymic generation of excited states of oxygen on the induction of benzo(alpha)pyrene mono-oxygenase in liver cell cultures.

The photochemical generation of excited states of oxygen such as the superoxide ion(O-2) and singlet oxygen (1o2) by the mild illumination of culture medium containing riboflavin induces benzo(alpha)pyrene mono-oxygenase in 3 different cell lines derived from rat liver. Similar rates of O-2 generation can be produced by the action of xanthine oxidase on xanthine yet this system does not induce the mono-oxygenase. This result confirms that the mono-oxygenase induction is not mediated by O-2 is not mediated by O-2 and that 1O2 is the most likely candidate for stimulating the mono-oxygenase activity.     

Induction of aryl hydrocarbon hydroxylase by a light-driven superoxide generating system in liver cell culture

Aryl Hydrocarbon Hydroxylase activity can be increased in rat liver cell cultures by the generation of the superoxide ion (O₂^?) in the growth medium. This can be achieved by mild illumination of the cells in the presence of riboflavin and methionine. The increased activity that results can be prevented by Cycloheximide and appears to be a typical induction. It is suggested that superoxide generation within cells may be a common factor linking different microsomal enzyme inducers.     

Trophoblast estrogen synthetase stimulation by dibutyryl cyclic AMP and theophylline: increase in cytochrome P-450 content.

The inclusion of both dibutyryl cyclic AMP and theophylline in the culture medium of human malignant trophoblast cells (JAr line) for 72 hours results in an enhanced estrogen secretion through the increased specific activity of estrogen synthetase (aromatase), a cytochrome P-450 mono-oxygenase enzyme system. The data described here suggest that this increased aromatase activity is due to an increase in the concentration of only one component of the mono-oxygenase system, cytochrome P-450.     

Active oxygen-associated control of rice blast disease by riboflavin and roseoflavin

Exogenous riboflavin and its dimethylated amino(nor)-derivative roseoflavin were studied in their ability to protect susceptible rice plants from blast disease and to induce fungitoxicity mediated by active oxygen. Both compounds, either added to the inoculum (10 microg/ml) or to soil (40 mg/kg, two days prior to inoculation), induced disease resistance, i.e., diminished the frequency of compatible-type lesions on infected leaves, mainly at the expense of the appearance of hypersensitive spots. Leaf diffusates of untreated plants possessed a weak fungitoxicity that increased slightly after leaf infection or illumination of diffusate. The flavins added to inoculum, to soil, or to a collected diffusate augmented significantly the light-activated part of the diffusate toxicity. In some instances, the light-independent part was stimulated as well. The effect was not due to direct fungitoxicity of flavins as they alone did not interfere with spores regardless of illumination. Antioxidant reagents (superoxide dismutase, catalase, scavengers of hydroxyl radical, and the iron ion chelator desferrioxamine) protected spores from intoxication in almost all cases. This implies the involvement of active oxygen in the toxic and, probably, disease-controlling effects of the flavins. Roseoflavin was a better inducer of disease resistance than riboflavin but was similar in stimulation of diffusate toxicity. However, roseoflavin did not produce superoxide and exhibited only weak fungitoxicity if substituted for riboflavin in the well-known O2--generating model photosystem containing methionine. Therefore, the superoxide generation due to photo-oxidation of methionine or similar substrates is not the cause of the increase of leaf diffusate fungitoxicity and of disease resistance of plants supplied with roseoflavin. It is suggested that the rise in active oxygen production favors a forthcoming hypersensitive reaction, and both phenomena contribute to resistance induced by flavo-compounds. The light-driven activation of oxygen may be of interest as a mode of action of novel fungicides.     

Mitochondrial dysfunction mediated by quinone oxidation products of dopamine: Implications in dopamine cytotoxicity and pathogenesis of Parkinson's disease

The study has demonstrated that dopamine induces membrane depolarization and a loss of phosphorylation capacity in dose-dependent manner in isolated rat brain mitochondria during extended in vitro incubation and the phenomena are not prevented by oxyradical scavengers or metal chelators. Dopamine effects on brain mitochondria are, however, markedly prevented by reduced glutathione and N-acetyl cysteine and promoted by tyrosinase present in the incubation medium. The results imply that quinone oxidation products of dopamine are involved in mitochondrial damage under this condition. When PC12 cells are exposed to dopamine in varying concentrations (100-400 μM) for up to 24 h, a pronounced impairment of mitochondrial bio-energetic functions at several levels is observed along with a significant (nearly 40%) loss of cell viability with features of apoptotic nuclear changes and increased activities of caspase 3 and caspase 9 and all these effects of dopamine are remarkably prevented by N-acetyl cysteine. N-acetyl cysteine also blocks nearly completely the dopamine induced increase in reactive oxygen species production and the formation of quinoprotein adducts in mitochondrial fraction within PC12 cells and also the accumulation of quinone products in the culture medium. Clorgyline, an inhibitor of MAO-A, markedly decreases the formation of reactive oxygen species in PC12 cells upon dopamine exposure but has only mild protective actions against quinoprotein adduct formation, mitochondrial dysfunctions, cell death and caspase activation induced by dopamine. The results have indicated that quinone oxidation products and not reactive oxygen species are primarily involved in cytotoxic effects of dopamine and the mitochondrial impairment plays a central role in the latter process. The data have clear implications in the pathogenesis of Parkinson's disease.     

Induction of stress proteins in chicken embryo cells by low-level zinc contamination in amino acid-free media

It has been reported that chicken embryo cells deprived of exogenous amino acids for 4 hours synthesize stress (heat-shock) proteins. Herein, we show that amino acid deprivation is not sufficient to cause induction of stress proteins. Zinc contaminating a component of commercial cell culture medium used to prepare amino acid-free medium was an inducer in our cultures. In the absence of exogenous amino acids, the concentration of zinc ions needed for half-maximal induction of stress proteins was an order of magnitude lower than the dose required for cells in complete medium. Histidine and cystine, which have high affinities for zinc ions, were the amino acids most effective in blocking the induction of stress proteins by zinc. Problems posed by heavy metal ions in culture media and biologic fluids for searches for in vivo inducers of the cellular stress (heat shock) response are discussed.     

Vitamin B2-sensitised photooxidation of the ophthalmic drugs Timolol and Pindolol: kinetics and mechanism

The kinetics and mechanistic aspects of the riboflavin-photosensitised oxidation of the topically administrable ophthalmic drugs Timolol (Tim) and Pindolol (Pin) were investigated in water-MeOH (9:1, v/v) solution employing light of wavelength > 400 nm. riboflavin, belonging to the vitamin B(2) complex, is a known human endogenous photosensitiser. The irradiation of riboflavin in the presence of ophthalmic drugs triggers a complex picture of competitive reactions which produces the photodegradation of both the drugs and the pigment itself. The mechanism was elucidated employing stationary photolysis, polarographic detection of dissolved oxygen, stationary and time-resolved fluorescence spectroscopy, and laser flash photolysis. Ophthalmic drugs quench riboflavin-excited singlet and triplet states. From the quenching of excited triplet riboflavin, the semireduced form of the pigment is generated, through an electron transfer process from the drug, with the subsequent production of superoxide anion radical (O(2)(*-)) by reaction with dissolved molecular oxygen. Through the interaction of dissolved oxygen with excited triplet riboflavin, the species singlet oxygen (O(2)((1)Delta(g))) is also generated to a lesser extent. Both O(2)(*-) and O(2)((1)Delta(g)) induce photodegradation of ophthalmic drugs, Tim being approximately 3-fold more easily photooxidisable than Pin, as estimated by oxygen consumption experiments.     

Oxidation of terfenadine by Streptomyces platensis: Influence of culture medium on metabolite formation

The biotransformation of terfenadine into a primary alcohol, hydroxyterfenadine, followed by its oxidation to an acid, fexofenadine, was investigated using Streptomyces platensis cells. Time-courses of metabolite formation were established, and the results underlined the modulation of the alcohol to acid formation ratio according to culture conditions. Optimization of the hydroxylation step (pH, temperature, culture medium composition) led to the preparation of hydroxyterfenadine with a good yield (51%) using cells grown in culture medium without soybean peptone. In contrast, when incubations were performed with cells cultured in a medium containing soybean peptone, the alcohol to acid formation ratio decreased. The efficiency of the conversion to fexofenadine was shown to depend on the age of the cells, thus suggesting the induction of an oxidative activity. Both the hydroxylation reaction and the following two-oxidation steps leading to the acid seemed to depend on oxygen.     

The induction of allophanate lyase during the vegetative cell cycle in light-synchronized cultures of Chlamydomonas reinhardi.

Allophanate lyase can be induced by urea or acetamide 20-40-fold within 4 h in NH4 + -deprived cultures of Chlamydomonas reinhardi. In light-synchronized cultures, allophanate lyase induction appeared to be limited to the light phase of the cell cycle, provided that culture samples were induced under ongoing illumination conditions (i.e. light induction of light phase cells and dark induction of dark phase cells). However, when culture samples were induced under constant light conditions this cell cycle pattern was abolished. Light was found to be required for allophanate lyase induction and this was shown to be due, in part, to the light requirement for inducer uptake. The relationship between allophanate lyase induction and gametogenesis is discussed.     

Oxidation of terfenadine by Streptomyces platensis: Influence of culture medium on metabolite formation

The biotransformation of terfenadine into a primary alcohol, hydroxyterfenadine, followed by its oxidation to an acid, fexofenadine, was investigated using Streptomyces platensis cells. Time-courses of metabolite formation were established, and the results underlined the modulation of the alcohol to acid formation ratio according to culture conditions. Optimization of the hydroxylation step (pH, temperature, culture medium composition) led to the preparation of hydroxyterfenadine with a good yield (51%) using cells grown in culture medium without soybean peptone. In contrast, when incubations were performed with cells cultured in a medium containing soybean peptone, the alcohol to acid formation ratio decreased. The efficiency of the conversion to fexofenadine was shown to depend on the age of the cells, thus suggesting the induction of an oxidative activity. Both the hydroxylation reaction and the following two-oxidation steps leading to the acid seemed to depend on oxygen.     

Feedback inhibition of hepatic DNA synthesis

alpha-Hexachlorocyclohexane (alpha-HCH) and some other xenobiotic inducers were used to elicit adaptive increases in mono-oxygenase activity, size and DNA content of rat liver. After elimination of the inducers, organ size and mono-oxygenase activity returned to normal whereas the DNA content of the liver remained increased. Upon renewed treatment with an inducer the adaptive responses uncoupled. While mono-oxygenase induction and liver enlargement did occur, DNA replication was largely suppressed. These findings show that in the hyperplastic state the liver is resistant to stimulation of DNA synthesis by the inducers. It is concluded that the DNA content of the liver (or the number of liver cells) is controlled by a feedback system which monitors an excess of DNA (cells) and suppresses cell replication if the content of DNA exceeds the normal level. Organ mass has little, if any, effect on the operation of this feedback system.     

FEEDBACK INHIBITION OF HEPATIC DNA SYNTHESIS

α-Hexachlorocyclohexane (α-HCH) and some other xenobiotic inducers were used to elicit adaptive increases in mono-oxygenase activity, size and DNA content of rat liver. After elimination of the inducers, organ size and mono-oxygenase activity returned to normal whereas the DNA content of the liver remained increased. Upon renewed treatment with an inducer the adaptive responses uncoupled. While mono-oxygenase induction and liver enlargement did occur, DNA replication was largely suppressed. These findings show that in the hyperplastic state the liver is resistant to stimulation of DNA synthesis by the inducers. It is concluded that the DNA content of the liver (or the number of liver cells) is controlled by a feedback system which monitors an excess of DNA (cells) and suppresses cell replication if the content of DNA exceeds the normal level. Organ mass has little, if any, effect on the operation of this feedback system.     

The induction of allophanate lyase during the vegetative cell cycle in light-synchronized cultures of Chlamydomonas reinhardi

Allophanate lyase can be induced by urea or acetamied 20–40-fold within 4 h in NH₄⁺-deprived cultures of Chlamydomonas reinhardi. In light-synchronized cultures, allophanate lyase induction appeared to be limited to the light phase of the cell cycle, provided that culture samples were induced under ongoing illumination conditions (i.e. light induction of light phase cells and dark induction of dark phase cells). However, when culture samples were induced under constant light conditions this cell cycle pattern was abolished. Light was found to be required for allophanate lyase induction and this was shown to be due, in part, to light requirement for inducer uptake. The relationship between allophanate lyase induction and gametogenesis is discussed.     

Lipid and lipoprotein oxidation: basic mechanisms and unresolved questions in vivo

Abstract

The kinetics and mechanistic aspects of the riboflavin-photosensitised oxidation of the topically administrable ophthalmic drugs Timolol (Tim) and Pindolol (Pin) were investigated in water–MeOH (9:1, v/v) solution employing light of wavelength > 400 nm. riboflavin, belonging to the vitamin B₂ complex, is a known human endogenous photosensitiser. The irradiation of riboflavin in the presence of ophthalmic drugs triggers a complex picture of competitive reactions which produces the photodegradation of both the drugs and the pigment itself. The mechanism was elucidated employing stationary photolysis, polarographic detection of dissolved oxygen, stationary and time-resolved fluorescence spectroscopy, and laser flash photolysis. Ophthalmic drugs quench riboflavin-excited singlet and triplet states. From the quenching of excited triplet riboflavin, the semireduced form of the pigment is generated, through an electron transfer process from the drug, with the subsequent production of superoxide anion radical (O₂^?–) by reaction with dissolved molecular oxygen. Through the interaction of dissolved oxygen with excited triplet riboflavin, the species singlet oxygen (O₂(¹Δ_g)) is also generated to a lesser extent. Both O₂^?– and O₂(¹Δ_g) induce photodegradation of ophthalmic drugs, Tim being ~3-fold more easily photooxidisable than Pin, as estimated by oxygen consumption experiments.     

Induction of sister chromatid exchanges in human cells by fluorescent light.

The Brd-U differential staining technique was utilized to examine the induction of sister-chromatid exchanges (SCE) by fluorescent ligt in human fetal lung fibroblasts (IMR-90). Exposure of these cells in media to fluorescent light resulted in an increase in SCE frequencies from a background level of 8.5 SCE/cell to 20.5 SCE/cell. Cellular replication kinetics were also inhibited by fluorescent light exposure. Exposure of cells to fluorescent light in phosphate buffered saline (PBS) resulted in a two-fold increase in SCE levels and incresed inhibition of cell replication, indicating that culture media may have a protective effect. Determinations of SCE frequencies with blocking filters indicated that the fluorescent light wavelengths responsible for SCE induction were in the near-ultraviolet spectrum between 300 and 390 nm. Culturing cell sin media that had been exposed to fluorescent light resulted in a significant increase in SCE levels, 14.5 ± 1.5 vs. 7.5 ± 0.65, demonstrating the contribution of media photoproducts to SCE induction. The role of media photoproducts was further reinforced by finding a significant decline in fluorescent light induced SCE in cells cultured in medium deficient in three known photosensitizers (phenol red, tetracycline and riboflavin) for 2–3 weeks prior to exposure.Since SCE have been shown to be a sensitive indicator of DNA damage, these results indicate that fluorescent light can induce genetic damage in human cells. These findings are also of importance to investigators culturing cells in laboratories with fluorescent illumination.     

A promising action of riboflavin as a mediator of leukaemia cell death

Besides having a pivotal biological function as a component of coenzymes, riboflavin appears a promissing antitumoral agent, but the underlying molecular mechanism remains unclear. In this work, we demonstrate that irradiated riboflavin, when applied at μM concentrations, induces an orderly sequence of signaling events finally leading to leukemia cell death. The molecular mechanism involved is dependent on the activation of caspase 8 caused by overexpression of Fas and FasL and also on mitochondrial amplification mechanisms, involving the stimulation of ceramide production by sphingomyelinase and ceramide synthase. The activation of this cascade led to an inhibition of mitogen activated protein kinases: JNK, MEK and ERK and survival mediators (PKB and IAP1), upregulation of the proapoptotic Bcl2 member Bax and downregulation of cell cycle progression regulators. Importantly, induction of apoptosis by irradiated riboflavin was leukaemia cell specific, as normal human lymphocytes did not respond to the compound with cell death. Our data indicate that riboflavin selectively activates Fas cascade and also constitutes a death receptor-engaged drug without harmful side effects in normal cells, bolstering the case for using this compound as a novel avenue for combating cancerous disease.     

Low density lipoprotein cytotoxicity induced by free radical peroxidation of lipid

Low density lipoprotein (LDL) has been reported to be injurious or toxic to cells in vitro. This injurious effect is, in some instances, due to oxidation of the lipid moiety of the lipoprotein. The objectives of this study were to determine if the oxidation rendering the lipoprotein toxic to human skin fibroblasts occurred by free radical mechanisms, and if so, which of the common free radical oxygen species were involved. The selective free radical blockers or scavengers employed included superoxide dismutase for superoxide, catalase for hydrogen peroxide, dimethylfuran for singlet molecular oxygen, and mannitol for hydroxyl radical. The presence during lipoprotein preparation of general free radical scavengers (vitamin E, butylated hydroxytoluene) or the divalent cation chelator ethylenediamine tetraacetic acid prevented the formation of cytotoxic low density lipoprotein, while the simultaneous presence of superoxide dismutase and catalase partially inhibited its formation. The results indicate that superoxide and/or hydrogen peroxide are involved in the formation of the toxic LDL lipid. The toxic action of oxidized LDL could not be prevented by inclusion of antioxidants in the culture medium, indicating that an oxidized lipid was responsible for cell injury rather than free radicals generated in culture by the action of oxidized LDL. Three separate assays for cell injury (enumeration of attached cells, cell loss of lactate dehydrogenase into the culture medium, and trypan blue uptake) indicated a sequence of events in which the fibroblasts are injured, die, and then detach.     

Interaction of molecular oxygen with the donor side of photosystem II after destruction of the water-oxidizing complex

Photosystem II (PSII) is a pigment-protein complex of thylakoid membrane of higher plants, algae, and cyanobacteria where light energy is used for oxidation of water and reduction of plastoquinone. Light-dependent reactions (generation of excited states of pigments, electron transfer, water oxidation) taking place in PSII can lead to the formation of reactive oxygen species. In this review attention is focused on the problem of interaction of molecular oxygen with the donor site of PSII, where after the removal of manganese from the water-oxidizing complex illumination induces formation of long-lived states (P680^+· and TyrZ^·) capable of oxidizing surrounding organic molecules to form radicals.     

Effect of riboflavie and cyanide on blue light induction of betacyanin formation in Amamnthus caudatus seedlings

The role of phyto chrome and flavins in blue light induction of betacyanin formation was studied in etiolated, three-day-old Amaranthus caudatus L. seedlings, using the criterion of far-red reversibility and exogenously applied riboflavin and KCN. The effect of riboflavin was studied using high fluence rate blue light (42.7 :nmol m⁻²s⁻¹nm⁻¹ at 450 nm). When present in the incubation medium during illumination, riboflavin promoted the far-red reversibility with short light treatments and suppressed the inductive action of continuous illumiaation. If added after light treatments, it promoted betacyanin formation. The filtration of blue light through the riboflavin solution caused profound changes in light quality without affecting the far-red reversibility after 30 mm illumination. The effect of 1 mM KCN was tested with 70'% lower fluence of blue light. Cyanide caused the suppression of the inductive effect with 5 min blue light, which was accompanied by an enhancement of betacyanin induction by the terminal far-red light pulse. With 30 min blue light, however, it caused the appearance of far-red reversibility. The inductive effect of continuous blue illumination was slightly promoted by this Inhibitor. These results demonstrate that the effect of blue light on the pbyto chrome system is complex, whereas the physiological (inductive) action of the flavin triplet state is limited to low fluence, short blue light treatments.