Spectroscopic evidence of interaction between 2-allyl-2-isopropylacetamide and cytochrome P-450 of rat liver microsomes

2-allyl-2-isopropylacetamide (AIA) causes marked induction of heme synthesis in rats and other species, degrades cytochrome P-450 in the presence of NADPH and causes experimental porphyria. Using difference spectroscopy we sought evidence of an interaction between AIA and P-450 in microsomes prepared from rat liver. AIA alone caused small and variable changes in the spectral properties of liver microsomes but markedly inhibited the Type I spectral change due to hexobarbitone. Phenobarbitone exhibited behaviour qualitatively similar to AIA. It is concluded that AIA binds to cytochrome P-450 without much altering its spectral properties but in such a way as to prevent the change induced by the Type I substrate hexobarbitone.     

A possible association of EMID2 polymorphisms with aspirin hypersensitivity in asthma

Aspirin-intolerant asthma (AIA) is an asthma phenotype characterized by the development of bronchoconstriction following ingestion of aspirin. Despite the well-defined pathological trigger, the underlying mechanisms of AIA are still unclear. With the biophysical characteristics of the human EMI domain-containing protein 2 (EMID2) gene in relation to the extracellular matrix deposition and epithelial-mesenchymal transition as pivotal characteristics of airway remodeling in asthma, we hypothesized that genetic polymorphisms of EMID2 might affect the development of AIA. In this study, the allelic associations of 49 single-nucleotide polymorphisms (SNPs) of the human EMID2 gene were evaluated from 163 AIA patients and 429 aspirin-tolerant asthma (ATA) subjects as controls in a Korean population. Logistic analysis showed that five SNPs (P?=?0.01-0.04, but P (corr)?>?0.05) and EMID2_BL2_ht2 haplotype (unique to the minor alleles of rs4727494 and rs13233066; P?=?0.02; P (corr)?=?0.02) were significantly associated with AIA. More interestingly, regression analysis of the decline of forced expiratory volume in one second (FEV(1)) by aspirin provocation revealed that 10 SNPs (P?=?0.003-0.04) and four relevant haplotypes (P?=?0.002-0.02) were significantly associated with the fall rate of FEV(1) by aspirin provocation, indicating that genetic polymorphisms of EMID2 could cause meaningful deficits in the upper and lower airways among AIA patients. These findings provide evidence that EMID2 may be a susceptible genetic factor for aspirin hypersensitivity among asthmatics in Korean population.     

Drug metabolizing enzyme system and heme pathway in hepatocarcinogenesis

Chemically induced and spontaneous liver tumors share some metabolic alterations. The decline in hemoprotein levels during hepatocarcinogenesis may result from a diminution of the intracellular heme pool. To elucidate if the onset of the pre-initiation stage alters the natural regulation mechanism of heme pathway, animals were fed with p-dimethylaminoazobenzene (DAB) and treated or not with 2-allylisopropylacetamide (AIA). The induction of 6-Aminolevulinic acid synthase (ALA-S) activity and the diminution in microsomal heme oxygenase (MHO) did not change when DAB fed animals were treated with AIA. Cytochrome P-450 (P-450) levels and glutathione S-transferase activity were increased in all the groups tested. Tryptophan pyrrolase, sulphatase and beta-glucuronidase activities were altered in DAB fed animals but AIA treatment did not produce any effect. Changes in drug metabolizing enzymes in livers of DAB fed animals could be the result of a primary deregulation of heme metabolism. These results give additional support to our hypothesis about a mechanism for the onset of hepatocarcinogenesis.     

Evidence for the in vitro metabolism of allylisopropylacetamide to reactive intermediates. Mechanistic studies with oxygen-18

Metabolism of allylisopropylacetamide (AIA) (1) in microsomal preparations from phenobarbital-pretreated rats is shown to proceed by way of three cytochrome P-450-dependent pathways: (i) aliphatic (C-3') hydroxylation, (ii) allylic (C-3) hydroxylation and (iii) olefin oxidation. The latter represents the major route of biotransformation and leads ultimately to the formation of the gamma-butyrolactone 2. In order to elucidate the mechanism by which AIA is converted to this gamma-lactone, and to gain information on the nature of chemically reactive intermediates in the process, the metabolism of AIA to 2 was investigated in 18O2 or H218O and the pattern of label incorporated into the product was determined by gas chromatography/mass spectrometry (GC/MS). The results support the formation of AIA epoxide as an initial product of olefin oxidation and indicate that this species undergoes rapid intramolecular rearrangement to a protonated iminolactone which, in turn, is hydrolysed to the stable gamma-lactone. On the other hand, the 'dihydrodiol' metabolite of AIA, which would be expected to result from direct hydrolysis of AIA epoxide, was not detected in incubation products and, furthermore, the 18O labeling data specifically exclude the possibility that it served as a precursor of 2. It may be concluded, therefore, that AIA epoxide and the protonated iminolactone to which it gives rise represent reactive intermediates in the oxidation of AIA which may play a key role in the alkylation of certain cellular constituents which accompanies metabolism of AIA by liver enzymes.     

Fibroblast growth factor-2 determines severity of joint disease in adjuvant-induced arthritis in rats.

Rheumatoid arthritis (RA), a systemic inflammatory disease of unknown etiology, mainly affects synovial joints. Although angiogenic growth factors, including fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF), may play a critical role in the development and progression of RA joint disease, little information is now available regarding their exact role in initiation and/or progression of RA. In this study, we show that both polypeptides were up-regulated in the rat joint synovial tissue of an adjuvant-induced model of arthritis (AIA), as well as human subjects with RA. FGF-2 overexpression via Sendai virus-mediated gene transfer significantly worsened clinical symptoms and signs of rat AIA, including hind paw swelling and radiological bone destruction, as well as histological findings based on inflammatory reaction, synovial angiogenesis, pannus formation, and osteocartilaginous destruction, associated with up-regulation of endogenous VEGF. FGF-2 gene transfer to non-AIA joints was without effect. These findings suggested that FGF-2 modulated disease progression, but did not affect initiation. Reverse experiments using anti-FGF-2-neutralizing rabbit IgG attenuated clinical symptoms and histopathological abnormalities of AIA joints. To our knowledge, this is the first report indicating direct in vivo evidence of disease-modulatory effects of FGF-2 in AIA, as probably associated with endogenous VEGF function. FGF-2 may prove to be a possible therapeutic target to treat subjects with RA.     

Microsomal prostaglandin E synthase-1 is a major terminal synthase that is selectively up-regulated during cyclooxygenase-2-dependent prostaglandin E2 production in the rat adjuvant-induced arthritis model

To better define the role of the various prostanoid synthases in the adjuvant-induced arthritis (AIA) model, we have determined the temporal expression of the inducible PGE synthase (mPGES-1), mPGES-2, the cytosolic PGES (cPGES/p23), and prostacyclin synthase, and compared with that of cyclooxygenase-1 (COX-1) and COX-2. The profile of induction of mPGES-1 (50- to 80-fold) in the primary paw was similar to that of COX-2 by both RNA and protein analysis. Quantitative PCR analysis indicated that induction of mPGES-1 at day 15 was within 2-fold that of COX-2. Increased PGES activity was measurable in membrane preparations of inflamed paws, and the activity was inhibitable by MK-886 to >or=90% with a potency similar to that of recombinant rat mPGES-1 (IC(50) = 2.4 microM). The RNA of the newly described mPGES-2 decreased by 2- to 3-fold in primary paws between days 1 and 15 postadjuvant. The cPGES/p23 and COX-1 were induced during AIA, but at much lower levels (2- to 6-fold) than mPGES-1, with the peak of cPGES/p23 expression occurring later than that of COX-2 and PGE(2) production. Prostacyclin (measured as 6-keto-PGF(1alpha)) was transiently elevated on day 1, and prostacyclin synthase was down-regulated at the RNA level after day 3, suggesting a diminished role of prostacyclin during the maintenance of chronic inflammation in the rat AIA. These results show that mPGES-1 is up-regulated throughout the development of AIA and suggest that it plays a major role in the elevated production of PGE(2) in this model.     

Ramipril attenuates lipid peroxidation and cardiac fibrosis in an experimental model of rheumatoid arthritis

Introduction

Recent studies revealed that co-morbidity and mortality due to cardiovascular disease are increased in patients with rheumatoid arthritis (RA) but little is known about factors involved in these manifestations. This study aimed at characterizing the impact of arthritis on oxidative stress status and tissue fibrosis in the heart of rats with adjuvant-induced arthritis (AIA).

Methods

AIA was induced with complete Freund's adjuvant in female Lewis rats. Animals were treated by oral administration of vehicle or angiotensin-converting enzyme inhibitor ramipril (10 mg/kg/day) for 28 days, beginning 1 day after arthritis induction. Isolated adult cardiomyocytes were exposed to 10 μM 4-hydroxynonenal (HNE) for 24 hours in the presence or absence of 10 μM ramipril.

Results

Compared to controls, AIA rats showed significant 55 and 30% increase of 4-HNE/protein adducts in serum and left ventricular (LV) tissues, respectively. Cardiac mitochondrial NADP+-isocitrate dehydrogenase (mNADP-ICDH) activity decreased by 25% in AIA rats without any changes in its protein and mRNA expression. The loss of mNADP-ICDH activity was correlated with enhanced accumulation of HNE/mNADP-ICDH adducts as well as with decrease of glutathione and NADPH. Angiotensin II type 1 receptor (AT₁R) expression and tissue fibrosis were induced in LV tissues from AIA rats. In isolated cardiomyocytes, HNE significantly decreased mNADP-ICDH activity and enhanced type I collagen and connective tissue growth factor expression. The oral administration of ramipril significantly reduced HNE and AT₁R levels and restored mNADP-ICDH activity and redox status in LV tissues of AIA rats. The protective effects of this drug were also evident from the decrease in arthritis scoring and inflammatory markers.

Conclusion

Collectively, our findings disclosed that AIA induced oxidative stress and fibrosis in the heart. The fact that ramipril attenuates inflammation, oxidative stress and tissue fibrosis may provide a novel strategy to prevent heart diseases in RA.     

2-Deoxy-D-glucose and 6-aminonicotinamide-mediated Nrf2 down regulation leads to radiosensitization of malignant cells via abrogation of GSH-mediated defense

Enhanced level of nuclear erythroid-related factor-2 (Nrf2) has been associated with cancer chemo/radioresistance. Therefore, the role of Nrf2 in radiosensitization of malignant cells induced by a combination of 2-deoxy-D-Glucose (2-DG) and 6-aminonicotinamide (6-AN) was investigated. Two established human malignant cells lines namely KB (head and neck squamous carcinoma) and BMG-1 (cerebral glioma) were used. Following treatment with a combination of 2-DG (5 mM) and 6-AN (5 μM), irradiated (2Gy) KB and BMG-1 cells were assessed for protein level of Nrf2, Keap1 and γ-glutamylcysteine synthetase (γ-GCS) by western blotting and mRNA expression of γ-GCS, glutathione reductase (GR) and glutathione peroxidase (GPx1) by RT-PCR at 24 hours post treatment. A significant decrease in the level of Nrf2 with a concomitant increase in Keap1 was observed in both the irradiated malignant cells at 24 hours following treatment with combination (2-DG + 6-AN). Down regulation of γ-GCS, GR and GPx1 at 24 hours following treatment with combination (2-DG + 6-AN) resulted in abrogation of glutathione (GSH)-mediated defense in both the irradiated malignant cells. Eventual accumulation of ROS led to radiosensitization of both the malignant cells. These results indicate that deregulated Nrf2-Keap1 signalling leads to the radiosensitization of malignant cells due to abrogated glutathione defense. Metabolic modification-mediated down regulation of Nfr2 and its downstream signalling may have a potential of improving tumour radiotherapy.     

Shift of the Cellular Oxidation-Reduction Potential in Neural Cells Expressing Bcl-2

Abstract: Expression of the protooncogene bcl-2 inhibits both apoptotic and in some cases necrotic cell death in many cell types, including neural cells, and in response to a wide variety of inducers. The mechanism by which the Bcl-2 protein acts to prevent cell death remains elusive. One mechanism by which Bcl-2 has been proposed to act is by decreasing the net cellular generation of reactive oxygen species. To evaluate this proposal, we measured activities of antioxidant enzymes as well as levels of glutathione and pyridine nucleotides in control and bcl-2 transfectants in two different neural cell lines—rat pheochromocytoma PC12 and the hypothalamic GnRH cell line GT1-7. Both neural cell lines overexpressing bcl-2 had elevated total glutathione levels when compared with control transfectants. The ratios of oxidized glutathione to total glutathione in PC12 and GT1-7 cells overexpressing bcl-2 were significantly reduced. In addition, the NAD⁺/NADH ratio of bcl-2 -expressing PC12 and GT1-7 cells was two- to threefold less than that of control cell lines. GT1-7 cells overexpressing bcl-2 had the same level of glutathione peroxidase, catalase, superoxide dismutase, and glutathione reductase activities as control cells. PC12 cells overexpressing bcl-2 had a twofold increase in superoxide dismutase and catalase activity when compared with matched control transfected cells. The levels of glutathione peroxidase and glutathione reductase in PC12 cells overexpressing bcl-2 were similar to those of control cells. These results indicate that the overexpression of bcl-2 shifts the cellular redox potential to a more reduced state, without consistently affecting the major cellular antioxidant enzymes.     

A comparison between the 2N and 4N HCl acid-insoluble ash methods for digestibility trials in horses

The digestibility of horse feeds and rations can be determined using different techniques such as calculations based on the chemical composition, in vivo or in vitro methods. The marker methods overcome difficulties like discomfort for the animals and longer experimental times encountered using the ingesta/egesta method. In field conditions, a natural indigestible marker like acid-insoluble ash (AIA), with no changes in the normal ration, could be a very useful tool for digestibility trials. A group of six standardbred horses was used in a set of seven apparent digestibility trials. The diets were based on a first-cut meadow hay added to three different cereals (barley for trials 1 and 2; oats for trials 3 and 5 and corn for trials 6 and 7), the hay : concentrate ratio being 60 : 40 or 70 : 30 on a dry matter basis. Feedstuffs and faeces were analysed to determine the AIA content, using 2N HCl or 4N HCl technique. No differences about AIA concentration were found between the two methods for means and accuracy in each diet. Digestion coefficients for each diet did not differ with AIA method, even if in some trials interfering factors consistently lowered the overall values. Consequently, the AIA 2N HCl can be considered the easier and cheaper method to state apparent digestibility in field conditions, and a good tool for digestibility trials in horses fed hay-based diets.     

Monoarticular antigen-induced arthritis leads to pronounced bilateral upregulation of the expression of neurokinin 1 and bradykinin 2 receptors in dorsal root ganglion neurons of rats

This study describes the upregulation of neurokinin 1 and bradykinin 2 receptors in dorsal root ganglion (DRG) neurons in the course of antigen-induced arthritis (AIA) in the rat knee. In the acute phase of AIA, which was characterized by pronounced hyperalgesia, there was a substantial bilateral increase in the proportion of lumbar DRG neurons that express neurokinin 1 receptors (activated by substance P) and bradykinin 2 receptors. In the chronic phase the upregulation of bradykinin 2 receptors persisted on the side of inflammation. The increase in the receptor expression is relevant for the generation of acute and chronic inflammatory pain.     

Exacerbation of antigen-induced arthritis in IFN-gamma-deficient mice as a result of unrestricted IL-17 response

Proinflammatory Th1 responses are believed to be involved in the induction and perpetuation of rheumatoid arthritis. However, the role of IFN-gamma, the major cytokine produced by Th1 cells, is still incompletely defined. In the present study, we investigated the effects of IFN-gamma deficiency (IFN-gamma(-/-)) on the course of experimental murine Ag-induced arthritis (AIA). In the acute stage of disease, IFN-gamma(-/-) AIA mice showed significantly increased inflammatory responses compared with wild-type C57BL/6 AIA mice, i.e., exacerbated joint swelling, increased delayed-type hypersensitivity reaction, and increased histopathological scores of arthritis. Intraarticular administration of exogenous IFN-gamma at induction of AIA significantly suppressed these acute aggravation effects. Stimulated cells isolated from lymph nodes and spleen of IFN-gamma(-/-) AIA mice showed increased production of IL-2, IL-4, IL-5, IL-6, but most prominently of IL-17. These elevations were paralleled by decreased humoral immune responses, with low serum levels of total and Ag-specific IgG (IgG1, IgG2a(b), IgG2b, IgG3). At immunohistology, the knee joints of IFN-gamma(-/-) AIA mice showed massive neutrophil granulocyte infiltration. Treatment with mAbs neutralizing IL-17 diminished the acute inflammation. In vitro, Th cell expansion and production of IL-17 upon restimulation were effectively and dose dependently inhibited by IFN-gamma. These results clearly demonstrate that IFN-gamma has anti-inflammatory properties during the initial phase of AIA, and indicate that IFN-gamma deficiency exerts disease-promoting effects, preferentially via IL-17-modulated pathways.     

Angiotensin II type 2 receptor correlates with therapeutic effects of losartan in rats with adjuvant‐induced arthritis

The angiotensin II type 1 receptor (AT1R) blocker losartan ameliorates rheumatoid arthritis (RA) in an experimental model. In RA, AT2R mainly opposes AT1R, but the mechanism by which this occurs still remains obscure. In the present study, we investigated the role of AT2R in the treatment of rats with adjuvant-induced arthritis (AIA) by losartan. Adjuvant-induced arthritis rats were treated with losartan (5, 10 and 15 mg/kg) and methotrexate (MTX; 0.5 mg/kg) in vivo from day 14 to day 28. Arthritis was evaluated by the arthritis index and histological examination. Angiotensin II, tumour necrosis factor-α, and VEGF levels were examined by ELISA. The expression of AT1R and AT2R was detected by western blot and immunohistochemistry analysis. After stimulation with interleukin-1β in vitro, the effects of the AT2R agonist {"type":"entrez-protein","attrs":{"text":"CGP42112","term_id":"874777115","term_text":"CGP42112"}}CGP42112 (10⁻⁸–10⁻⁵ M) on the chemotaxis of monocytes induced by 10% foetal calf serum (FCS) were analysed by using Transwell assay. Subsequently, the therapeutic effects of {"type":"entrez-protein","attrs":{"text":"CGP42112","term_id":"874777115","term_text":"CGP42112"}}CGP42112 (5, 10 and 20 μg/kg) were evaluated in vivo by intra-articular injection in AIA rats. After treatment with losartan, the down-regulation of AT1R expression and up-regulation of AT2R expression in the spleen and synovium of AIA rats correlated positively with reduction in the polyarthritis index. Treatment with {"type":"entrez-protein","attrs":{"text":"CGP42112","term_id":"874777115","term_text":"CGP42112"}}CGP42112 inhibited the chemotaxis of AIA monocytes in vitro, possibly because of the up-regulation of AT2R expression. Intra-articular injection with {"type":"entrez-protein","attrs":{"text":"CGP42112","term_id":"874777115","term_text":"CGP42112"}}CGP42112 (10 and 20 μg/kg) ameliorated the arthritis index and histological signs of arthritis. In summary, the present study strongly suggests that the up-regulation of AT2R might be an additional mechanism by which losartan exerts its therapeutic effects in AIA rats.     

Reduction of mitochondrial protein mitoNEET [2Fe–2S] clusters by human glutathione reductase

The human mitochondrial outer membrane protein mitoNEET is a newly discovered target of the type 2 diabetes drug pioglitazone. Structurally, mitoNEET is a homodimer with each monomer containing an N-terminal transmembrane α helix tethered to the mitochondrial outer membrane and a C-terminal cytosolic domain hosting a redox-active [2Fe–2S] cluster. Genetic studies have shown that mitoNEET has a central role in regulating energy metabolism in mitochondria. However, the specific function of mitoNEET remains largely elusive. Here we find that the mitoNEET [2Fe–2S] clusters can be efficiently reduced by Escherichia coli thioredoxin reductase and glutathione reductase in an NADPH-dependent reaction. Purified human glutathione reductase has the same activity as E. coli thioredoxin reductase and glutathione reductase to reduce the mitoNEET [2Fe–2S] clusters. However, rat thioredoxin reductase, a human thioredoxin reductase homolog that contains selenocysteine in the catalytic center, has very little or no activity to reduce the mitoNEET [2Fe–2S] clusters. N-ethylmaleimide, a potent thiol modifier, completely inhibits human glutathione reductase from reducing the mitoNEET [2Fe–2S] clusters, indicating that the redox-active disulfide in the catalytic center of human glutathione reductase may be directly involved in reducing the mitoNEET [2Fe–2S] clusters. Additional studies reveal that the reduced mitoNEET [2Fe–2S] clusters in mouse heart cell extracts can be reversibly oxidized by hydrogen peroxide without disruption of the clusters, suggesting that the mitoNEET [2Fe–2S] clusters may undergo redox transition to regulate energy metabolism in mitochondria in response to oxidative signals.     

Properties and functions of glutathione reductase in plants

The assay and in vitro characterization of glutathione reductase (EC 1.6.4.2) is discussed. In vivo the H₂O₂-scavenging system in chloroplasts is the best documented role of reduced glutathione and glutathione reductase in plants. Similarly, redaction of H₂O₂, outside of the chloroplasts, requires glutathione and glutathione reductase; but the pathway, in terms of intermediates, is controversial. The notion that biological stress frequently causes cellular oxidation has lead to the suggestion that glutathione and glutathione reductase may play a role in stress resistance or tolerance mechanisms. The changes in glutathione reductase levels in response to low temperature, oxidative stress and drought are discussed.     

Regulation of the activity of glyceraldehyde 3-phosphate dehydrogenase by glutathione and H2O2

The activity lost during storage of a solution of muscle glyceraldehyde 3-phosphate dehydrogenase was rapidly restored on adding a thiol compound, but not arsenite or azide. On treatment with H₂O₂, the enzyme was partially inactivated and complete loss of activity occurred in the presence of glutathione. Samples of the enzyme pretreated with glutathione followed by removal of the thiol compound by filtration on a Sephadex column showed both full activity and its complete loss on adding H₂O₂, in the absence of added glutathione. Most of the activity was restored when the H₂O₂-inactivated enzyme was incubated with glutathione (25mM) or dithiothreitol (5mM) whereas arsenite or azide were partly effective and ascorbate was ineffective. The need for incubation for a long time with a strong reducing agent for restoration of activity suggests that the oxidized group (disulfide or sulfenate) must be in a masked state in the H₂O₂-inactivated enzyme. Analysis by SDS-PAGE gave evidence for the formation of a small quantity of glutathione-reversible disulfide-form of the enzyme. Circular dichroic spectra indicated a decrease in -helical content in the inactivated form of the enzyme. The evidence suggest that glutathione and H₂O₂ can regulate the active state of this enzyme.     

Redox modulation of tau and microtubule-associated protein-2 by the glutathione/glutaredoxin reductase system

Alterations in the redox status of proteins have been implicated in the pathology of several neurodegenerative diseases including Alzheimer's and Parkinson's. We report that peroxynitrite and H2O2-induced disulfides in the porcine brain microtubule-associated proteins tau and microtubule-associated protein-2 are substrates for the glutaredoxin reductase system composed of glutathione reductase, human or Escherichia coli glutaredoxin, reduced glutathione, and NADPH. Oxidation and reduction of cysteines in tau and microtubule-associated protein-2 were quantitated by monitoring the incorporation of 5-iodoacetamido-fluorescein, a thiol-specific labeling reagent. Reduction of disulfide bonds in the microtubule-associated proteins by the glutaredoxin reductase system restored their ability to promote the assembly of microtubules composed of purified porcine tubulin. Thiol-disulfide exchange between oxidized glutathione and the microtubule-associated proteins was detected by monitoring protein oxidation and was quantitated by measuring reduced glutathione by HPLC.     

Ultrasonic vocalization response elicited in adjuvant-induced arthritic rats as a useful method for evaluating analgesic drugs.

Adjuvant-induced arthritic (AIA) rats develop a severe chronic polyarthritis which shares some features in common with human rheumatoid arthritis. The purpose of the present study was to examine whether AIA rats emit ultrasonic vocalizations (USVs) when they are confronted with a healthy 'stimulus rat' in social interactions. We also examined the effects of three analgesic drugs (piroxicam, rofecoxib and ketoprofen) on USV responses using the same paradigm. In social interactions, AIA rats and intact controls emitted USVs in the 22-28 kHz range. Vocalization activities were significantly higher in AIA rats than those in intact controls. Moreover, the USVs of AIA rats were significantly inhibited by the three analgesic drugs. These results suggest that the USV responses elicited in AIA rats are useful for the evaluation of analgesic drugs.     

Decrease in hepatic cytochrome P-450 and catalase following allylisopropylacetamide: The effect of concomitant hemin administration

It is known that administration of allylisopropylacetamide (AIA) to rats increases δ-aminolevulinic acid synthetase (ALA-S), urine ascorbic acid excretion and decreases hepatic hemoproteins cytochrome P-450 and catalase. Hemin has been shown to inhibit induction of ALA-S by AIA. To investigate further the regulatory role of hemin, AIA was administered to rats over a 5 day period with and without hemin. Hemin did not prevent the decrease in P-450 or catalase caused by AIA but partially inhibited induction of ALA-S and the increase in ascorbic acid excretion. It is unlikely that the hemin is replacing endogenous heme in hemoprotein synthesis and we conclude that the role of hemin in suppressing induction phenomena that follow AIA treatment reflects a regulatory function not related to a role in hemoprotein synthesis.     

The redox state of the ascorbate-dehydroascorbate pair as a specific sensor of cell division in tobacco BY-2 cells

Summary The effects of ascorbate (ASC) and dehydroascorbate (DHA) on cell proliferation were examined in the tobacco Bright Yellow 2 (TBY-2) cell line to test the hypothesis that the ASC-DHA pair is a specific regulator of cell division. The hypothesis was tested by measuring the levels of ASC and DHA or another general redox pair, glutathione (GSH) and glutathione disulfide (GSSG), during the exponential-growth phase of TBY-2 cells. A peak in ASC, but not GSH, levels coincided with a peak in the mitotic index. Moreover, when the cells were enriched with ascorbate, a stimulation of cell division occurred whereas, when the cells were enriched with DHA, the mitotic index was reduced. In contrast, glutathione did not affect the mitotic-index peak during this exponential-growth phase. The data are consistent in showing that the ASC-DHA pair acts as a specific redox sensor which is part of the mechanism that regulates cell cycle progression in this cell line.