Rune Djurhuus

Ornithine decarboxylase (EC 4.1.1.17) assay based upon the retention of putrescine by a strong cation-exchange paper

Abstract A simple and direct method for the detection of ornithine decarboxylase (EC 4.1.1.17) activity is presented. It is based upon the selective binding of putrescine to Whatman P81 paper, a strong cation-exchanger, in the presence of 0.1 m NH 3 . The assay is easy to perform and has an advantage over the more frequently used CO 2 trapping methods which can yield spurious CO 2 formation due to the action of enzymes other than ornithine decarboxylase.

Low-dose exposure to alkylphenols adversely affects the sexual development of Atlantic cod (Gadus morhua): Acceleration of the onset of puberty and delayed seasonal gonad development in mature female cod

Produced water (PW), a by-product of the oil-production process, contains large amount of alkylphenols (APs) and other harmful oil compounds. In the last 20 years, there have been increasing concerns regarding the environmental impact of large increases in the amounts of PW released into the North Sea. We have previously shown that low levels of APs can induce disruption of the endocrine and reproductive systems of Atlantic cod (Gadus morhua). The aims of this follow-up study were to: (i) identify the lowest observable effect concentration of APs; (ii) study the effects of exposure to real PW, obtained from a North Sea oil-production platform; and (iii) study the biological mechanism of endocrine disruption in female cod. Fish were fed with feed paste containing several concentrations of four different APs (4-tert-butylphenol, 4-n-pentylphenol, 4-n-hexylphenol and 4-n-heptylphenol) or real PW for 20 weeks throughout the normal period of vitellogenesis in Atlantic cod from October to January. Male and female cod, exposed to AP and PW, were compared to unexposed fish and to fish fed paste containing 17β-oestradiol (E(2)). Approximately 60% of the females and 96% of the males in the unexposed groups were mature at the end of the experiment. Our results show that exposure to APs and E(2) have different effects depending on the developmental stage of the fish. We observed that juvenile females are advanced into puberty and maturation, while gonad development was delayed in both maturing females and males. The AP-exposed groups contained increased numbers of mature females, and significant differences between the untreated group and the AP-treated groups were seen down to a dose of 4 μg AP/kg body weight. In the high-dose AP and the E(2) exposed groups, all females matured and no juveniles were seen. These results suggest that AP-exposure can affect the timing of the onset of puberty in fish even at extremely low concentrations. Importantly, similar effects were not seen in the fish that were exposed to real PW.

Additive toxicity of limonene and 50% oxygen and the role of glutathione in detoxification in human lung cells

Limonene has many commercial applications and has been introduced as an environmentally acceptable solvent replacing halogenated hydrocarbons. Occupational exposure to limonene presumably occurs simultaneously with other chemicals including oxidative agents and may exert a heavy strain on cellular detoxifying capacity resulting in synergistic effects. The present study used oxygen as an example of an ubiquitous oxidative and radical forming agent and investigated the combination effects with limonene on human lung cells. Mechanistic information was gained by comparing the toxicity of limonene with a major oxidation product, limonene 1,2-epoxide, and by the involvement of glutathione in cellular detoxification. At cell culture conditions most similar to the in vivo situation oxygen did not increase the toxicity of limonene beyond an additive effect. The results further indicated that limonene 1,2-epoxide was not the active compound in limonene toxicity. Experimental evidence suggests that detoxification of limonene in human lung cells primarily occurs by mechanisms not involving the glutathione system and point to possible long-term effects of limonene exposure. The present knowledge indicates clearly that the mechanism of action of limonene on biological systems and particularly in combination with oxidative compounds still remains to be elucidated. In light of the frequent exposure of humans to such combinations further investigations into this issue are highly recommended.

A nonradioactive assay fo N5-methyltetrahydrofolate-homocysteine methyltransferase (methionine synthase) based on o-phthaldialdehyde derivatization of methionine and fluorescence detection

Abstract The enzyme N 5 -methyltetrahydrofolate-homocysteine methyltransferase (methionine synthase, EC 2.1.1.13) catalyzes the conversion of homocysteine to methionine in the presence of a reducing system. N 5 -Methyltetrahydrofolate serves as a methyl donor in this reaction. An assay for the enzyme is described, which is based on methionine quantitation by o -phthaldialdehyde (OPA) derivatization and reversed-phase liquid chromatography. The enzymatic reaction is linear for at least 120 min under reducing conditions (125 m m 2-mercaptoethanol) and running the assay below an oil layer. This reducing system does not interfere with formation of the methionine-OPA adduct, which is separated from interfering compounds and an internal standard (norvaline) by a mobile phase adjusted to pH 5.0. The inclusion of internal standard increases the precision of the assay and corrects for the variable fluorescence yield due to occasional inaccurate pH adjustment before the derivatization step. Norvaline was suitable for this purpose because it elutes close to methionine and is not a natural amino acid present in biological extracts. This nonradioactive assay for methionine synthase was evaluated by comparison with a conventional method based on isolation of radioactive methionine by anion-exchange chromatography and by determination of enzyme activity in extract from cultured cells and liver.

Simultaneous quantification of tetrahydrobiopterin, dihydrobiopterin, and biopterin by liquid chromatography coupled electrospray tandem mass spectrometry

A simple and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method was developed for the quantification of tetrahydrobiopterin (BH4), dihydrobiopterin (BH2), and biopterin (B) in human umbilical vein endothelial cells (HUVECs). Freshly prepared cell samples were treated with a mixture consisting of 0.2M trichloroacetic acid (TCA) and a cocktail of various antioxidants in order to precipitate proteins and other cellular components and to stabilize red/ox conditions in the lysates. Chromatography of the cell lysates was performed on a Poroshell 120 SB-C18 column (2.7μm, 150×2.1mm) using a stepwise gradient elution made from two mobile phases. Quantification was performed on a triple quadrupole mass spectrometer employing electrospray ionization with the operating conditions as multiple reaction monitoring (MRM) at positive ion mode. Total chromatographic run time was 23min. The method was validated for analysis in HUVECs, and the limits of quantification were 1nM for BH4 and BH2 and 2.5nM for B. Standard curves were linear in the concentration ranges of 1 to 100nM for BH4 and BH2 and 2.5 to 100nM for B. The current study reports a novel method for the simultaneous and direct quantification of BH4, BH2, and B in a single injection.

Perturbation of Homocysteine Metabolism by Pharmacological Agents in Experimental and Clinical Use

Homocysteine, a sulfur amino acid, was discovered over 50 years ago by DuVigneaud as the product of demethylation of methionine This discovery was followed by reports that homocysteine could support growth of animals fed diets deficient in either cysteine, methionine or choline (1).

Proposal on limits for chemical exposure in saturation divers' working atmosphere: the case of benzene.

Saturation diving is performed under extreme environmental conditions. The divers are confined to a limited space for several weeks under high environmental pressure and elevated oxygen partial pressure. At present, divers are protected against chemical exposure by standard exposure limits only adjusted for the increased exposure length, i.e. from 8 to 24 hours a day and from 5 to 7 days a week. The objective of the present study was to indicate a procedure for derivation of occupational exposure limits for saturation diving, termed hyperbaric exposure limits (HEL). Using benzene as an example, a procedure is described that includes identification of the latest key documents, extensive literature search with defined exclusion criteria for the literature retrieved. Hematotoxicity and leukemia were defined as the critical effects, and exposure limits based upon concentration and cumulative exposure data and corresponding risks of leukemia were calculated. Possible interactions of high pressure, elevated pO2, and continuous exposure have been assessed, and incorporated in a final suggestion of a HEL for benzene. The procedure should be applicable for other relevant chemicals in the divers’ breathing atmosphere. It is emphasized that the lack of interactions from pressure and oxygen indicated for benzene may be completely different for other chemicals.

Ornithine decarboxylase activity in foetal rat brain cells and in the mouse embryo fibroblasts C3H/10T1/2 CL8 cells: Differences in response to medium change and to the tumor promoter TPA

12-O-tetradecanoyl-phorbol-13-acetate (TPA) induced ornithine decarboxylase (ODC, EC 4.1.1.17) in normal, preneoplastic and malignant rat brain cells in culture, but treatment with phorbol, acetate or medium shift resulted in a similar response. Medium shift induced ODC activity in C3H/10T1/2 CL8 cells 4 and 12 hr after treatment. TPA induced only the 12 hr peak. ODC induction in C3H/10T1/2 CL8 cells was completely inhibited by cycloheximide and actinomycin D. Addition of alpha-amanitin abolished the 12 hr peak, but the TPA induced ODC activity was only partly inhibited. ODC induction by TPA was lower in C3H/10T1/2 CL8 cells initiated with 3-methyl-cholanthrene (MCA). ODC increased with TPA up to 10(-7) M and decreased at higher concentrations of TPA.