Isopropyl m-Chlorocarbanilate and Its Hydroxylated Metabolites: Their Effects on Cell Suspensions and Cell Divison in Soybean and Carrot

Hydroxylated metabolites of isopropyl m-chlorocarbanilate (chlorpropham) are found in intact soybean plants (Glycine max Merr.). The metabolites are isopropyl 2-hydroxy-5-chloro-carbanilate (2OH) and isopropyl 3-chloro-4-hydroxycarbanilate (4OH). The phytotoxicity of these metabolites and chlorpropham was tested in cell suspensions and roots of intact soybean seedlings and cell cultures of carrot (Daucus carota L.). The growth of soybean cell suspensions was inhibited with 50 μM chlorpropham. Ten μM chlorpropham usually slowed initial growth of the cultures while 5 μM and 0.1 μM chlorpropham had no effect. The 2OH and 4OH metabolites had no significant effect on dry weight over the same concentration range. Some metabolism of chlorpropham, 2OH and 4OH occurred during 6 and 48 h of incubation with soybean cells. The results are interpreted to mean that all three analogs penetrated into the cells, were metabolized, and some of the metabolites excreted back into the medium. Mitotic index studies of intact 3-day-old soybean roots showed that 2OH inhibited mitosis to a greater extent than chlorpropham, whereas 4OH produced only a slight and insignificant reduction compared to controls. Chlorpropham, 2OH and 4OH (at 50 μM) all reduced the growth of wild carrot cultures grown in the presence or absence of 2,4-D. Therefore, hydroxylation of chlorpropham at the 2′ or 4′ positions of the 5′ chlorinated benzene ring is not sufficient to render the compound nonphytotoxic in all plant systems.     

Increased placental concentrations of 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 in pregnant rats treated with human chorionic gonadotropin

Pregnant rats were injected intrajugularly with 2500 i.u. human chorionic gonadotropin (HCG) toward the end of gestation (days 18-19) and 7.0 pmoles of tritiated 25-hydroxyvitamin D3 [( 3H]25(OH)D3) the following day. They were sacrificed ten to 24 hours later. [3H]25(OH)D3 and the in vivo produced [3H]24,25-dihydroxyvitamin D3 [( 3H]24,25(OH)2D3) in lipid extracts from maternal serum, kidneys, placenta and fetal tissues were separated by Sephadex LH-20 chromatography, and high performance liquid chromatography (HPLC). HCG treatment of pregnant rats increased significantly 25(OH)D3 levels in the placenta and kidneys and 24,25(OH)2D3 level in the placenta. Fetal metabolites levels were unaffected by HCG treatment. Serum and kidney levels of 25(OH)D3 and 24,25(OH)2D3 in pregnant rats were significantly lower than in non-pregnant rats. Serum and kidney levels of both metabolites in non-pregnant female rats treated with HCG did not differ from the untreated controls. HCG may, therefore, be involved in regulation of fetoplacental vitamin D metabolism.     

Dual metabolic pathway of 25-hydroxyvitamin D3 catalyzed by human CYP24.

Human 25-hydroxyvitamin D3 (25(OH)D3) 24-hydroxylase (CYP24) cDNA was expressed in Escherichia coli, and its enzymatic and spectral properties were revealed. The reconstituted system containing the membrane fraction prepared from recombinant E. coli cells, adrenodoxin and adrenodoxin reductase was examined for the metabolism of 25(OH)D3, 1alpha,25(OH)2D3 and their related compounds. Human CYP24 demonstrated a remarkable metabolism consisting of both C-23 and C-24 hydroxylation pathways towards both 25(OH)D3 and 1alpha,25(OH)2D3, whereas rat CYP24 showed almost no C-23 hydroxylation pathway [Sakaki, T. Sawada, N. Nonaka, Y. Ohyama, Y. & Inouye, K. (1999) Eur. J. Biochem. 262, 43-48]. HPLC analysis and mass spectrometric analysis revealed that human CYP24 catalyzed all the steps of the C-23 hydroxylation pathway from 25(OH)D3 via 23S, 25(OH)2D3, 23S,25,26(OH)3D3 and 25(OH)D3-26,23-lactol to 25(OH)D3-26, 23-lactone in addition to the C-24 hydroxylation pathway from 25(OH)D3 via 24R,25(OH)2D3, 24-oxo-25(OH)D3, 24-oxo-23S,25(OH)2D3 to 24,25,26,27-tetranor-23(OH)D3. On 1alpha,25(OH)2D3 metabolism, similar results were observed. These results strongly suggest that the single enzyme human CYP24 is greatly responsible for the metabolism of both 25(OH)D3 and 1alpha,25(OH)2D3. We also succeeded in the coexpression of CYP24, adrenodoxin and NADPH-adrenodoxin reductase in E. coli. Addition of 25(OH)D3 to the recombinant E. coli cell culture yielded most of the metabolites in both the C-23 and C-24 hydroxylation pathways. Thus, the E. coli expression system for human CYP24 appears quite useful in predicting the metabolism of vitamin D analogs used as drugs.     

Application of engineered cytochrome P450 mutants as biocatalysts for the synthesis of benzylic and aromatic metabolites of fenamic acid NSAIDs

Cytochrome P450 BM3 mutants are promising biocatalysts for the production of drug metabolites. In the present study, the ability of cytochrome P450 BM3 mutants to produce oxidative metabolites of structurally related NSAIDs meclofenamic acid, mefenamic acid and tolfenamic acid was investigated. A library of engineered P450 BM3 mutants was screened with meclofenamic acid (1) to identify catalytically active and selective mutants. Three mono-hydroxylated metabolites were identified for 1. The hydroxylated products were confirmed by NMR analysis to be 3′-OH-methyl-meclofenamic acid (1a), 5-OH-meclofenamic acid (1b) and 4′-OH-meclofenamic acid (1c) which are human relevant metabolites. P450 BM3 variants containing V87I and V87F mutation showed high selectivity for benzylic and aromatic hydroxylation of 1 respectively. The applicability of these mutants to selectively hydroxylate structurally similar drugs such as mefenamic acid (2) and tolfenamic acid (3) was also investigated. The tested variants showed high total turnover numbers in the order of 4000–6000 and can be used as biocatalysts for preparative scale synthesis. Both 1 and 2 could undergo benzylic and aromatic hydroxylation by the P450 BM3 mutants, whereas 3 was hydroxylated only on aromatic rings. The P450 BM3 variant M11 V87F hydroxylated the aromatic ring at 4′ position of all three drugs tested with high regioselectivity. Reference metabolites produced by P450 BM3 mutants allowed the characterisation of activity and regioselectivity of metabolism of all three NSAIDs by thirteen recombinant human P450s. In conclusion, engineered P450 BM3 mutants that are capable of benzylic or aromatic hydroxylation of fenamic acid containing NSAIDs, with high selectivity and turnover numbers have been identified. This shows their potential use as a greener alternative for the generation of drug metabolites.     

Hepatic bile acid metabolism during early development revealed from the analysis of human fetal gallbladder bile

A detailed study of the qualitative and quantitative composition of bile acids in human fetal gallbladder bile is described. Bile was collected during early gestation (weeks 16-19) and analyzed by gas chromatography and mass spectrometry, fast atom bombardment ionization mass spectrometry, and high performance liquid chromatography. Bile acids were separated into different conjugate groups by chromatography on the lipophilic anion exchange gel, diethylaminohydroxypropyl Sephadex LH-20. Quantitatively more than 80% of the bile acids were secreted into bile conjugated to taurine. Unconjugated bile acids and glycine conjugates accounted for 5-10% of the total biliary bile acids. Bile acid sulfates were present only in trace amounts indicating that quantitatively sulfation is not an important pathway in bile acid metabolism during development. Total biliary bile acid concentrations were low (0.1-0.4 mM) when compared to reported values for adult bile (greater than 10 mM). Chenodeoxycholic acid was the major biliary bile acid and exceeded cholic acid concentrations by 1.43-fold indicating either a relative immaturity in 12 alpha-hydroxylase activity during early life or a dominance of alternative pathways for chenodeoxycholic acid synthesis. A relatively large proportion of the biliary bile acids comprised metabolites not found in adult bile. The presence of relatively high proportions of hyocholic acid (often greater than cholic acid) and several 1 beta-hydroxycholanoic acid isomers indicates that C-1 and C-6 hydroxylation are important pathways in bile acid synthesis during development. We describe, for the first time, evidence for the existence of a C-4 hydroxylation pathway in the metabolism of bile acids, which may be unique to early human development. Mass spectrometry was used to confirm the identification of 3 alpha,4 beta,7 alpha-trihydroxy-5 beta-cholanoic and 3 alpha,4 beta-dihydroxy-5 beta-cholanoic acids. Quantitatively, these C-4 hydroxylated bile acids accounted for 5-15% of the total biliary bile acids of the fetus, suggesting that C-4 hydroxylation is quantitatively an important pathway in the bile acid metabolism during early life.     

Novel metabolism of 1 alpha,25-dihydroxyvitamin D3 with C24-C25 bond cleavage catalyzed by human CYP24A1

Our previous study revealed that human CYP24A1 catalyzes a remarkable metabolism consisting of both C-23 and C-24 hydroxylation pathways that used both 25(OH)D(3) and 1alpha,25(OH)(2)D(3) as substrates, while rat CYP24A1 showed extreme predominance of the C-24 over C-23 hydroxylation pathway [Sakaki, T., Sawada, N., Komai, K., Shiozawa, S., Yamada, S., Yamamoto, K., Ohyama, Y. and Inouye, K. (2000) Eur. J. Biochem. 267, 6158-6165]. In this study, by using the Escherichia coli expression system for human CYP24A1, we identified 25,26,27-trinor-23-ene-D(3) and 25,26,27-trinor-23-ene-1alpha(OH)D(3) as novel metabolites of 25(OH)D(3) and 1alpha,25(OH)(2)D(3), respectively. These metabolites appear to be closely related to the C-23 hydroxylation pathway, because human CYP24A1 produces much more of these metabolites than does rat CYP24A1. We propose that the C(24)-C(25) bond cleavage occurs by a unique reaction mechanism including radical rearrangement. Namely, after hydrogen abstraction of the C-23 position of 1alpha,25(OH)(2)D(3), part of the substrate-radical intermediate is converted into 25,26,27-trinor-23-ene-1alpha(OH)D(3), while a major part of them is converted into 1alpha,23,25(OH)(3)D(3). Because the C(24)-C(25) bond cleavage abolishes the binding affinity of 1alpha,25(OH)D(3) for the vitamin D receptor, this reaction is quite effective for inactivation of 1alpha,25(OH)D(3).     

C-25 hydroxylation of 1alpha,24(R)-dihydroxyvitamin D3 is catalyzed by 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1): metabolism studies with human keratinocytes and rat recombinant CYP24A1

Recently, 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1) has been shown to catalyze not only hydroxylation at C-24 but also hydroxylations at C-23 and C-26 of the secosteroid hormone 1alpha, 25-dihydroxyvitamin D3 (1alpha,25(OH)2D3). It remains to be determined whether CYP24A1 has the ability to hydroxylate vitamin D3 compounds at C-25. 1alpha,24(R)-dihydroxyvitamin D3 (1alpha,24(R)(OH)2D3) is a non-25-hydroxylated synthetic vitamin D3 analog that is presently being used as an antipsoriatic drug. In the present study, we investigated the metabolism of 1alpha,24(R)(OH)2D3 in human keratinocytes in order to examine the ability of CYP24A1 to hydroxylate 1alpha,24(R)(OH)2D3 at C-25. The results indicated that keratinocytes metabolize 1alpha,24(R)(OH)2D3 into several previously known both 25-hydroxylated and non-25-hydroxylated metabolites along with two new metabolites, namely 1alpha,23,24(OH)3D3 and 1alpha,24(OH)2-23-oxo-D3. Production of the metabolites including the 25-hydroxylated ones was detectable only when CYP24A1 activity was induced in keratinocytes 1alpha,25(OH)2D3. This finding provided indirect evidence to indicate that CYP24A1 catalyzes C-25 hydroxylation of 1alpha,24(R)(OH)2D3. The final proof for this finding was obtained through our metabolism studies using highly purified recombinant rat CYP24A1 in a reconstituted system. Incubation of this system with 1alpha,24(R)(OH)2D3 resulted in the production of both 25-hydroxylated and non-25-hydroxylated metabolites. Thus, in our present study, we identified CYP24A1 as the main enzyme responsible for the metabolism of 1alpha,24(R)(OH)2D3 in human keratinocytes, and provided unequivocal evidence to indicate that the multicatalytic enzyme CYP24A1 has the ability to hydroxylate 1alpha,24(R)(OH)2D3 at C-25.     

Metabolism of 1α,25-dihydroxyvitamin D2 by human CYP24A1

The metabolism of 1α,25-dihydroxyvitamin D₂ (1α,25(OH)₂D₂) by human CYP24A1 was examined using the recombinant enzyme expressed in Escherichia coli cells. HPLC analysis revealed that human CYP24A1 produces at least 10 metabolites, while rat CYP24A1 produces only three metabolites, indicating a remarkable species-based difference in the CYP24A1-dependent metabolism of 1α,25(OH)₂D₂ between humans and rats. LC-MS analysis and periodate treatment of the metabolites strongly suggest that human CYP24A1 converts 1α,25(OH)₂D₂ to 1α,24,25,26(OH)₄D₂, 1α,24,25,28(OH)₄D₂, and 24-oxo-25,26,27-trinor-1α(OH)D₂ via 1α,24,25(OH)₃D₂. These results indicate that human CYP24A1 catalyzes the C₂₄-C₂₅ bond cleavage of 1α,24,25(OH)₂D₂, which is quite effective in the inactivation of the active form of vitamin D₂. The combination of hydroxylation at multiple sites and C-C bond cleavage could form a large number of metabolites. Our findings appear to be useful to predict the metabolism of vitamin D₂ and its analogs in the human body.     

Long-chain metabolites of vitamin E: Interference with lipotoxicity via lipid droplet associated protein PLIN2

The long-chain metabolites of vitamin E (LCM) emerge as a new class of regulatory metabolites and have been considered as the active compounds formed during vitamin E metabolism. The bioactivity of the LCM is comparable to the already established role of other fat-soluble vitamins. The biological modes of action of the LCM are far from being unraveled, but first insights pointed to distinct effects and suggested a specific receptor, which in turn lead to the aforementioned hypothesis. Here, a new facet on the interaction of LCM with foam cell formation of THP-1 macrophages is presented. We found reduced levels of mRNA and protein expression of lipid droplet associated protein PLIN2 by α-tocopherol (α-TOH), whereas the LCM and the saturated fatty acid, stearic acid, increased expression levels of PLIN2. In a lipotoxic setup (0–800?μM stearic acid and 0–100?μM α-TOH or 0–5?μM α-13′-COOH) differences in cellular viability were found. A reduced viability was observed for cells under co-treatment of α-TOH and stearic acid, whereas an increased viability for stearic acid incubation in combination with α-13′-COOH was observed. The striking similarity of PLIN2 expression levels and worsened or mitigated lipotoxicity, respectively, revealed a protective effect of PLIN2 on basal stearic acid-induced lipotoxic conditions in PLIN2 knockdown experiments. Based on our results, we conclude that α-13′-COOH protects cells from lipotoxicity, at least partially via PLIN2 regulation.Herewith another facet of LCM functionality was presented and their reputation as regulatory metabolites was further established.     

Channel induction by palytoxin in yeast cells expressing Na+,K+-ATPase or its chimera with sarco/endoplasmic reticulum Ca2+-ATPase

Palytoxin (PTX) induces a cation channel through interaction with Na(+),K(+)-ATPase. It is unclear how this action relates to the enzyme catalytic activity. We examined whether the action of PTX depends on the catalytic domain specific for Na(+),K(+)-ATPase. Wild-type Na(+),K(+)-ATPase alpha-subunit (NNN) or its chimera (NCN), in which the catalytic domain was replaced with that of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase, was co-expressed with beta-subunit in the yeast Saccharomyces cerevisiae. PTX (0.1-100 nM) increased K(+) efflux in NNN- or NCN-transfected cells to a similar degree but not in non-transfected cells. When ouabain-resistant NNN and NCN were expressed, PTX also increased K(+) efflux. Ouabain inhibited the effect of PTX in NNN or NCN cells but not in ouabain-resistant cells. These data suggest that the channel-forming action of PTX does not depend on the catalytic domain species.     

The metabolism of 3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholestan-26-oic acid into cholic: an enzyme assay using homogenates of human liver.

An enzyme assay was developed to measure the conversion of the bile acid precursor, 3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholestan-26-oic acid (THCA), into cholic acid using homogenates of human liver biopsies. The average rate of metabolism of THCA into cholic acid was found to be 3.9 +/- 0.5 (+/- 1 SD) pmoles of cholic acid formed/mg liver/minute in twelve normal liver biopsies. This assay system can be used to determine if the syndrome of neonatal cholestasis associated with a metabolic block in the conversion of THCA into cholic acid is transmitted as a genetic trait.     

Cell and species differences in metabolic activation of chemical carcinogens

Metabolic activation and DNA binding of aflatoxin B1 (AFB1), N-nitrosodimethylamine (DMN) and benzo[a]pyrene (B[a]P) were compared in human, rat and mouse hepatocytes and human pulmonary alveolar macrophages (PAM). The degree of carcinogen activation by hepatocytes and PAM was measured by cell-mediated mutagenesis assays in which co-cultivated Chinese hamster V79 cells were used to monitor mutagenic metabolites. Hepatocytes from human, mouse and rat metabolized DMN and released the active metabolites to induce either ouabain- or 6-thioguanine-resistant mutation. The mutation frequencies mediated by hepatocytes of the 3 animal species were approximately 3-9 mutants/10(5) survivors at a concentration of 0.2 mM DMN. The variations of radioactivity bound to liver cell DNA were relatively small in cultured mouse, rat, and human hepatocytes exposed to 14C label DMN (0.5 mM) and the binding values were in a range of 6-12 X 10(3) pmoles/mg DNA. However, rat hepatocytes were at least 10-fold more effective than either human or mouse hepatocytes in generating mutagenic metabolites of AFB1 and also had a much higher AFB1 metabolite DNA-binding value. The AFB1 DNA-binding levels were 4.1, 12-27 (range), 120 pmoles/mg DNA respectively in mouse, human, and rat liver cells following AFB1 (3.3 microM) exposure for 20 h. Hepatocytes from the 3 animal species were unable to mediate mutation in the presence of 4 microM B[a]P; PAM activated B[a]P and effectively mediated mutation in the co-cultivated V79 cells. In contrast to results with hepatocytes, PAM failed to generate enough mutagenic metabolites of AFB1 (3.3 microM) and the mediation of mutations was seen only at very high concentration of DMN (80 mM). The genotoxic effects of the 3 carcinogens on hepatocytes from different species in vitro were in agreement with the in vivo animal experiments in that mice are relatively resistant to AFB1 carcinogenesis whereas rats are sensitive; B[a]P is not effective as a complete liver carcinogen in adult rat and mouse whereas DMN induces liver cancer.     

Defensive and secondary metabolism in plant tissue cultures,with special reference to nicotinamide,glutathione and oxidative stress

The purpose of the presentation is to interconnect and illuminate certain parts of metabolism regarding stress signalling and defensive functions, including secondary metabolism in intact plants and plant tissue cultures. Increased cell/tissue levels of reactive oxygen species like H₂O₂, O₂ ^- and ·OH and the metabolism of glutathione, are linked to defensive/secondary metabolism and tissue differentiation. Special attention is paid to nicotinamide. A hypothetical role of nicotinamide and its metabolites as stress signals is also put forward especially in connection with hypomethylation of DNA. A role of DNA hypomethylation, as a link between various types of stressors and the induction of plant devensive metabolism, is discussed. We suggest that nicotinamide or nicotinamide based substances may be of value within biotechnology for the production of valuable substances as well as for plant protection.Abbreviations BSO buthionine sulfoximine - CHS chalcone synthase - GSH reduced glutathione - GSSG oxidized glutathione - INA isonicotinic acid - NIC nicotinamide - PADPRP poly(ADP-ribose)polymerase - PAL phenylalanine ammonia-lyase - SA salicylic acid - SAM S-adenosylmethionine - SSB single strand breakage     

Determination of salicylate hydroxylation products as an in vivo oxidative stress marker

The in vivo measurement of highly reactive free radicals, such as the z.rad OH radical, is very difficult. New specific markers, which are based on the ability of z.rad OH to attack the benzene rings of aromatic molecules, are currently under investigation. The produced hydroxylated compounds can be measured directly. In vivo, radical metabolism of salicylic acid produces two main hydroxylated derivatives (2,3- and 2,5-dihydroxybenzoic acids). The latter acid can be also produced by enzymatic pathways through the cytochrome P-450 system, while the former acid is reported to be solely formed by direct hydroxyl radical attack. Therefore, measurement of 2, 3-DHBA, following oral administration of the drug acetyl salicylate, could be proposed for assessment of oxidative stress in vivo. In this paper, a sensitive method for the identification and quantification of hydroxylation products from the reaction of z. rad OH with salicylate in vivo is presented. It employs a high performance liquid chromatography and electrochemical detection system. A detection limit of < 1 pmol for the hydroxylation products has been achieved with linear response over at least five orders of magnitude. Using this technique, we measured plasma levels of 2,3- and 2,5-DHBA dihydroxylated derivatives and salicylic acid and determined the ratios following administration of 1 g acetyl salicylate in 20 healthy subjects.     

Deoxyribonucleoside triphosphate pools in Neurospora crassa: effects of histidine and hydroxyurea

An effective HPLC method for detecting deoxyribonucleoside triphosphates in hyphae from the fungus Neurospora crass has been developed. In rapidly growing cells the nucleotide levels vary from 11.8 pmoles/μg DNA for dGTP to 24.2 pmoles/μg DNA for dTTP. These levels fall by approximately one half in stationary-phase cultures but the ration of each pool to dGTP remains the same. The dNTP pools in conidia are at least 5-fold lower than in rapidly growing cells. The pool sizes are the same in static and shaking cultures. When the ribonucleotide reductase inhibitor, hydroxyurea (30 mM), is added to rapidly growing cultures, DNA synthesis is stopped and the dGTP pool is reduced by 39%, while the size of the other poolds remains the same. In the presence of 11 mM histadine, DNA synthesis is also stopped and the size of the dGTP pool reduced by 46% while the deoxypyrimidine pools are somewhat increased. This suggests that the toxicity of excess histidine in Neurospora may be due to its ability to interact with the ribonucleotide reductase, inactivating the enzyme. Histidine may react with free radical at the active sites, as does hydroxyurea.     

Deoxyribunucleoside triphosphate pools in Neurospora crassa: effects of histadine and hydroxyurea

An effective HPLC method for detecting deoxyribonucleoside triphosphates in hyphae from the fungus Neurospora crass has been developed. In rapidly growing cells the nucleotide levels vary from 11.8 pmoles/μg DNA for dGTP to 24.2 pmoles/μg DNA for dTTP. These levels fall by approximately one half in stationary-phase cultures but the ration of each pool to dGTP remains the same. The dNTP pools in conidia are at least 5-fold lower than in rapidly growing cells. The pool sizes are the same in static and shaking cultures. When the ribonucleotide reductase inhibitor, hydroxyurea (30 mM), is added to rapidly growing cultures, DNA synthesis is stopped and the dGTP pool is reduced by 39%, while the size of the other poolds remains the same. In the presence of 11 mM histadine, DNA synthesis is also stopped and the size of the dGTP pool reduced by 46% while the deoxypyrimidine pools are somewhat increased. This suggests that the toxicity of excess histidine in Neurospora may be due to its ability to interact with the ribonucleotide reductase, inactivating the enzyme. Histidine may react with free radical at the active sites, as does hydroxyurea.     

Collagen biosynthesis by human skin fibroblasts. III. The effects of ascorbic acid on procollagen production and prolyl hydroxylase activity

Human skin fibroblasts were cultured under conditions optimized for collagen synthesis, and the effects of ascorbic acid on procollagen production, proline hydroxylation and the activity of prolyl hydroxylase were examined in cultures. the results indicated that addition of ascorbic acid to confluent monolayer cultures of adult human skin fibroblasts markedly increased the amount of [3H]hydroxyproline synthesized. Ascorbic acid, however, did not increase the synthesis of 3H-labeled collagenous polypeptides assayed independently of hydroxylation of proline residues, nor did it affect the amount of prolyl hydroxylase detectable by an in vitro enzyme assay. Also long-term cultures of the cells or initiation of fibroblast cultures in the presence of ascorbic acid did not lead to an apparent selection of a cell population which might be abnormally responsive to ascorbic acid. Thus, ascorbic acid appears to have one primary action on the synthesis of procollagen by cultured human skin fibroblasts: it is necessary for synthesis of hydroxyproline, and consequently for proper triple helix formation and secretion of procollagen.     

Effects on DHEA levels by estrogen in rat astrocytes and CNS co-cultures via the regulation of CYP7B1-mediated metabolism

The neurosteroid dehydroepiandrosterone (DHEA) is formed locally in the CNS and has been implicated in several processes essential for CNS function, including control of neuronal survival. An important metabolic pathway for DHEA in the CNS involves the steroid hydroxylase CYP7B1. In previous studies, CYP7B1 was identified as a target for estrogen regulation in cells of kidney and liver. In the current study, we examined effects of estrogens on CYP7B1-mediated metabolism of DHEA in primary cultures of rat astrocytes and co-cultures of rat CNS cells. Astrocytes, which interact with neurons in several ways, are important for brain neurosteroidogenesis. We found that estradiol significantly suppressed CYP7B1-mediated DHEA hydroxylation in primary mixed CNS cultures from fetal and newborn rats. Also, CYP7B1-mediated DHEA hydroxylation and CYP7B1 mRNA were markedly suppressed by estrogen in primary cultures of rat astrocytes. Interestingly, diarylpropionitrile, a well-known agonist of estrogen receptor β, also suppressed CYP7B1-mediated hydroxylation of DHEA. Several previous studies have reported neuroprotective effects of estrogens. The current data indicate that one of the mechanisms whereby estrogen can exert protective effects in the CNS may involve increase of the levels of DHEA by suppression of its metabolism.     

Salivary thiocyanate/nitrite inhibits hydroxylation of 2-hydroxybenzoic acid induced by hydrogen peroxide/Fe(II) systems under acidic conditions: possibility of thiocyanate/nitrite-dependent scavenging of hydroxyl radical in the stomach

Formation of OH radicals in the stomach is possible by Fenton-type reactions, as gastric juice contains ascorbic acid (AA), iron ions and H2O2. An objective of the present study is to elucidate the effects of salivary SCN- and NO2- on the hydroxylation of salicylic acid which was induced by H2O2/Fe(II) and AA/H2O2/Fe(II) systems. Thiocyanate ion inhibited the hydroxylation of salicylic acid by the above systems in acidic buffer solutions and in acidified saliva. The inhibition by SCN- was deduced to be due to SCN- -dependent scavenging of OH radicals. Nitrite ion could enhance the SCN- -dependent inhibition of the hydroxylation induced by AA/H2O2/Fe(II) systems. The enhancement was suggested to be due to scavenging of OH radicals by NO which was formed by the reactions among AA, HNO2 and SCN- contained in the reaction mixture. The concentrations of SCN- and NO2-, which were effective for the inhibition, were in ranges of their normal salivary concentrations. These results suggest that salivary SCN- can cooperate with NO2- to protect stomach from OH radicals formed by AA/H2O2/Fe(II) systems under acidic conditions.     

Isolation,identification and determination of sulfadiazine and its hydroxy metabolites and conjugates from man and Rhesus monkey by high-performance liquid chromatography

The following metabolites of sulfadiazine (S) were isolated from monkey urine by preparative HPLC: 5-hydroxysulfadiazine (5OH), 4-hydroxysulfadiazine (4OH) and the glucuronide (5OHgluc) and sulfate conjugate of 5OH (5OHsulf). The compounds were identified by NMR, mass and infrared spectrometry and hydrolysis by β-glucuronidase. The analysis of S, the hydroxymetabolites (4OH, 5OH) and conjugates N₄-acetylsulfadiazine (N₄), 5OHgluc and 5OHsulf in human and monkey plasma and urine samples was performed using reversed-phase gradient HPLC with UV detection. In plasma, S and N₄ could be detected in high concentrations, whereas the other metabolites were present in only minute concentrations. In urine, S, the metabolites and conjugates were present. The limit of quantification of the compounds in plasma varies between 0.2 and 0.6 μg/ml (S 0.31, N₄ 0.40, 4OH 0.20, 5OH 0.37, 5OHgluc 0.33 and 5OHsulf 0.57 μg/ml). In urine it varies between 0.6 and 1.1 μg/ml (S 0.75, N₄ 0.80, 4OH 0.60, 5OH 0.80, 5OHgluc 0.80 and 5OHsulf 1.1 μg/ml). The method was applied to studies with healthy human subjects and Rhesus monkeys. The metabolites 5OH, 5OHgluc and 5OHsulf were present in Rhesus monkey and not in man. Preliminary results of studies of metabolism and pharmacokinetics in Rhesus monkey and man are presented.