Arsenic, mode of action at biologically plausible low doses : what are the implications for low dose cancer risk?

Arsenic is an established human carcinogen. However, there has been much controversy about the shape of the arsenic response curve, particularly at low doses. This controversy has been exacerbated by the fact that the  mechanism(s) of arsenic carcinogenesis are still unclear and because there are few satisfactory animal models for arsenic-induced carcinogenesis. Recent epidemiological studies have shown that the relative risk for cancer among populations exposed to ≤60 ppb As in their drinking water is often lower than the risk for the unexposed control population. We have found that treatment of human keratinocyte and fibroblast cells with 0.1 to 1 μM arsenite (As^III) also produces a low dose protective effect against oxidative stress and DNA damage. This response includes increased transcription, protein levels and enzyme activity of several base excision repair genes, including DNA polymerase β and DNA ligase I. At higher concentrations (> 10 μM), As induces down-regulation of DNA repair, oxidative DNA damage and apoptosis. This low dose adaptive (protective) response by a toxic agent is known as hormesis and is characteristic of many agents that induce oxidative stress. A mechanistic model for arsenic carcinogenesis based on these data would predict that the low dose risk for carcinogenesis should be sub-linear. The threshold dose where toxicity outweighs protection is hard to predict based on in vitro dose response data, but might be estimated if one could determine the form (metabolite) and concentration of arsenic responsible for changes in gene regulation in the target tissues.

A whole body, in vivo, fatty A\acid balance method to quantify PUFA metabolism (desaturation, elongation and beta-oxidation)

Currently there are several contrasting methods utilized for estimating elongation and desaturation of fatty acids and their general metabolism. The majority of these methods involve an ex vivo approach, requiring expensive and sophisticated equipment, likely to result in considerable variation in enzyme activity between and within species. In the present paper we introduce a further development of the whole-body fatty acid balance method for the estimation of the elongation and desaturation of fatty acids. This method though receiving considerable attention because of its simplicity and reliability has yet to be presented in detail. Theoretically, the whole-body fatty acid balance method can potentially be applied to any organism and requires little more than a gas chromatography unit for fatty acid analysis and elementary calculations. As such in this paper we attempt to spell out in detail the theoretical basis and the methods of application drawing specific examples. Using the present method it is possible to measure the fate of individual fatty acids towards desaturation, elongation and oxidation and calculate the elongase, Δ-6 desaturase and Δ-5 desaturase activities.

MMP2 and MMP9 plasma levels as markers of bone remodeling : a study on young male tennis players

Introduction
Our goal is to study the effects of tennis practice in pre-pubescent boys on bone remodeling, by means of enzyme activity involved in balance of matrix remodeling (MMP2 and MMP9).
Results
Mineral bone density has been found higher in the dominant arm (P < 0.0001) as well as MMP2 and MMP9 levels in plasma (P < 0.05).
Conclusion
Tennis practice in children increases bone remodeling, which can be assessed by MMP dosage, in addition of densitometry technique.

In vitro effect of arsenical compounds on glutathione-related enzymes

The mechanism of arsenic toxicity is believed to be due to the ability of arsenite (AsIII) to bind protein thiols. Glutathione (GSH) is the most abundant cellular thiol, and both GSH and GSH-related enzymes are important antioxidants that play an important role in the detoxification of arsenic and other carcinogens. The effect of arsenic on the activity of a variety of enzymes that use GSH has been determined using purified preparations of glutathione reductase (GR) from yeast and bovine glutathione peroxidase (GPx) and equine glutathione S-transferase (GST). The effect on enzyme activity of increasing concentrations (from 1 μM to 100 mM) of commercial sodium arsenite (AsIII) and sodium arsenate (AsV) and a prepared arsenic(III)−glutathione complex [AsIII(GS)3] and methylarsenous diiodide (CH3AsIII) has been examined.

A role for skeletal muscle stearoyl-CoA desaturase 1 in control of thermogenesis

An enhanced metabolic efficiency for accelerating the recovery of fat mass (or catch-up fat) is a characteristic feature of body weight regulation after weight loss or growth retardation and is the outcome of an "adipose-specific" suppression of thermogenesis, i.e., a feedback control system in which signals from the depleted adipose tissue fat stores exert a suppressive effect on thermogenesis. Using a previously described rat model of semistarvation-refeeding in which catch-up fat results from suppressed thermogenesis per se, we report here that the gene expression of stearoyl-coenzyme A desaturase 1 (SCD1) is elevated in skeletal muscle after 2 wk of semistarvation and remains elevated in parallel to the phase of suppressed thermogenesis favoring catch-up fat during refeeding. These elevations in the SCD1 transcript are skeletal muscle specific and are associated with elevations in microsomal ^9 desaturase enzyme activity, in the ^9 desaturation index, and in the relative content of SCD1-derived monounsaturates in several lipid fractions extracted from skeletal muscle. An elevated skeletal muscle SCD1, by desaturating the products of de novo lipogenesis and diverting them away from mitochondrial oxidation, would inhibit substrate cycling between de novo lipogenesis and lipid oxidation, thereby leading to a state of suppressed thermogenesis that regulates the body’s fat stores.—Mainieri, D., Summermatter, S., Seydoux, J., Montani, J. P., Rusconi, S., Russell, A. P., Boss, O., Buchala, A. J., Dulloo, A. G. A role for skeletal muscle stearoyl-CoA desaturase 1 in control of thermogenesis.

Increased probiotic yogurt or resistant starch intake does not affect isoflavone bioavailability in subjects consuming a high soy diet

Objective - Probiotics and prebiotics that affect gut microflora balance and its associated enzyme activity may contribute to interindividual variation in isoflavone absorption after soy intake, possibly enhancing isoflavone bioavailability. This study examined the effects of the consumption of bioactive yogurt (a probiotic) or resistant starch (a known prebiotic) in combination with high soy intake on soy isoflavone bioavailability.

Methods - Using a crossover design, chronic soy consumption was compared with soy plus probiotic yogurt or resistant starch in older male and postmenopausal females (n = 31). Isoflavone bioavailability was assessed at the beginning and end of each 5-wk dietary period by sampling plasma and urine after a standardized soy meal.

Results - Chronic soy intake did not significantly affect plasma or urinary isoflavones after the soy meal and there were no significant effects of probiotic or resistant starch treatment. However, there were trends for increased circulating plasma daidzein and genistein after the probiotic treatment and for increased plasma daidzein and genistein 24 h after soy intake with resistant starch treatment. Neither treatment induced or increased equol production, although there was a trend for increased plasma equol in “equol-positive” subjects (n = 12) after probiotic treatment.

Conclusion - The weak or absence of effects of probiotic yogurt or resistant starch supplement to a chronic soy diet suggests that gut microflora were not modified in a manner that significantly affected isoflavone bioavailability or metabolism.

Effect of nitric oxide synthase inhibition on mitochondrial biogenesis in rat skeletal muscle

The purpose of this study was to determine whether nitric oxide synthase (NOS) inhibition decreased basal and exercise-induced skeletal muscle mitochondrial biogenesis. Male Sprague-Dawley rats were assigned to one of four treatment groups: NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME, ingested for 2 days in drinking water, 1 mg/ml) followed by acute exercise, no L-NAME ingestion and acute exercise, rest plus L-NAME, and rest without L-NAME. The exercised rats ran on a treadmill for 53 ± 2 min and were then killed 4 h later. NOS inhibition significantly (P < 0.05; main effect) decreased basal peroxisome proliferator-activated receptor-{gamma} coactivator 1beta (PGC-1beta) mRNA levels and tended (P = 0.08) to decrease mtTFA mRNA levels in the soleus, but not the extensor digitorum longus (EDL) muscle. This coincided with significantly reduced basal levels of cytochrome c oxidase (COX) I and COX IV mRNA, COX IV protein and COX enzyme activity following NOS inhibition in the soleus, but not the EDL muscle. NOS inhibition had no effect on citrate synthase or beta-hydroxyacyl CoA dehydrogenase activity, or cytochrome c protein abundance in the soleus or EDL. NOS inhibition did not reduce the exercise-induced increase in peroxisome proliferator-activated receptor-{gamma} coactivator 1{alpha} (PGC-1{alpha}) mRNA in the soleus or EDL. In conclusion, inhibition of NOS appears to decrease some aspects of the mitochondrial respiratory chain in the soleus under basal conditions, but does not attenuate exercise-induced mitochondrial biogenesis in the soleus or in the EDL.

Purification and characterization of naringinase from a newly isolated strain of Aspergillus niger 1344 for the transformation of flavonoids

An extracellular naringinase (an enzyme complex consisting of α-L-rhamnosidase and β-D-glucosidase activity, EC 3.2.1.40) that hydrolyses naringin (a trihydroxy flavonoid) for the production of rhamnose and glucose was purified from the culture filtrate of Aspergillus niger 1344. The enzyme was purified 38-fold by ammonium sulphate precipitation, ion exchange and gel filtration chromatography with an overall recovery of 19% with a specific activity of 867 units per mg of protein. The molecular mass of the purified enzyme was estimated to be about 168 kDa by gel filtration chromatography on a Sephadex G-200 column and the molecular mass of the subunits was estimated to be 85 kDa by sodium dodecyl sulphate-Polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme had an optimum pH of 4.0 and temperature of 50 °C, respectively. The naringinase was stable at 37 °C for 72 h, whereas at 40 °C the enzyme showed 50% inactivation after 96 h of incubation. Hg2+, SDS, p-chloromercuribenzoate, Cu2+ and Mn2+ completely inhibited the enzyme activity at a concentration of 2.5–10 mM, whereas, Ca2+, Co2+ and Mg2+ showed very little inactivation even at high concentrations (10–100 mM). The enzyme activity was strongly inhibited by rhamnose, the end product of naringin hydrolysis. The enzyme activity was accelerated by Mg2+ and remained stable for one year after storage at −20 °C. The purified enzyme preparation successfully hydrolysed naringin and rutin, but not hesperidin.

Partial purification and characterization of limonoate dehydrogenase from Rhodococcus fascians for the degradation of limonin

An extracellular limonoate dehydrogenase was purified 10-fold from a cell-free extract of Rhodococcus fascians by ammonium sulfate precipitation, dialysis, and ultrafiltration. This purified dehydrogenase catalyzed the
conversion of limonoate to 17-dehydrolimonoate. The enzyme showed optimum activity at pH 8.0 and 40oC, with K_m value of 0.9 µM, and requires Zn ions and sulfhydryl groups for catalytic action. The enzyme activity was inhibited by Hg²⁺ and NaN₃ ions. The degradation of limonin (66%) in Kinnow mandarin juice was successfully demonstrated with partially
purified limonoate dehydrogenase. With scale-up preparation of limonoate dehydrogenase, a successful debittering operation of fruit juices appears feasible.

Partial purification and characterization of exoinulinase from kluyveromyces marxianus YS-1 for preparation of high-fructose syrup

An extracellular exoinulinase( ^{2, 1}- ß- D fructan fructanohydrolase, EC 3.2.1.7), which catalyzes the hydrolysis of inulin into fructose and glucose, was purified 23.5-fold by ethanol precipitation, followed by Sephadex G-100 gel permeation from a cell-free extract of Kluyveromyces marxianus YS-1. The partially purified enzyme exhibited considerable activity between pH 5 to 6, with an optimum pH of 5.5, while it remained stable(100%) for 3 h at the optimum temperature of 50º c. Mn²⁺ and Ca²⁺ produced a 2A-fold and 1.2-fold enhancement in enzyme activity, whereas Hg²⁺ and Ag²⁺  completely inhibited the inulinase. A preparation of the partially purified enzyme effectively hydrolyzed inulin, sucrose, and raffinose, yet no activity was found with starch, lactose, and maltose. The enzyme preparation was then successfully used to hydrolyze pure inulin and raw inulin from Asparagus racemosus for the preparation of a high-fructose syrup. In a batch system, the exoinulinase hydrolyzed 84.8% of the pure inulin and 86.7% of the raw Asparagus racemosus inulin, where fructose represented 43.6mg/ml and 41.3mg/ml, respectively.

植物CytP450和抗氧化酶对土壤菲、芘暴露的生态毒理响应

Maize (Zea mays L.) for the tested plants, meadow brown soil as the soil tested in order to microsomal cytochrome P450 content, superoxide dismutase (SOD), catalase (CAT) and peroxidase enzyme (POD) activity of indicators, the soil phenanthrene and pyrene in response to exposure to eco-toxicological studies. The results show that phenanthrene, pyrene exposure can cause detoxification of plant metabolism and antioxidant defense system of the stress response, caused varying degrees of detoxification of plant metabolism and changes in antioxidant capacity. P450 enzyme activity and low concentrations of phenanthrene and pyrene in a single - relevant exposure concentration (r = 0.834, P <0.01), and phenanthrene and pyrene exposure concentration was negatively correlated compound, saying that Ming Fei, pyrene compound exposed to lead detoxification metabolism of a reduced ability to detoxify the metabolism of plants have synergistic toxic effects; SOD activity and phenanthrene and pyrene in a single exposure concentration was negatively correlated, CAT activity and phenanthrene and pyrene in a single - exposure concentration was positively correlated, POD activity and water solubility of the Philippines positively correlated with the total concentration of pyrene in a negative correlation. SOD, CAT and POD activity and phenanthrene and pyrene were positively related to the concentration of compound exposure, saying that Ming Fei, pyrene complex degree of exposure to lead to reduced oxidative damage, oxidative damage of plants with antagonistic effects .

N3-substituted xanthines as irreversible adenosine receptor antagonists

8-Cyclopentyl-3-(3-(4-fluorosulfonylbenzoyl)oxy)propyl-propylxanthine (44, FSCPX) has been reported to exhibit potent and selective irreversible antagonism of the A1 adenosine receptor when using in vitro biological preparations. However, FSCPX (44) suffers from cleavage of the ester linkage separating the reactive 4-(fluorosulfonyl)phenyl moiety from the xanthine pharmacophore when used in in vivo biological preparations or preparations containing significant enzyme activity, presumably by esterases. Cleavage of the ester linkage renders FSCPX (44) inactive in terms of irreversible receptor binding. In order to obtain an irreversible A1 adenosine receptor antagonist with improved stability, and to further elucidate the effects of linker structure on pharmacological characteristics, several FSCPX (44) analogues incorporating the chemoreactive 4-(fluorosulfonyl)phenyl moiety were targeted, where the labile ester linkage has been replaced by more stable functionalites. In particular, ether, alkyl, amide and ketone linkers were targeted, where the length of the alkyl chain was varied from between one to five atoms. Synthesis of the target compounds was achieved via direct attachment of the N-3 substituent to the xanthine. These compounds were then tested for their biological activity at the A1 adenosine receptor via their ability to irreversibly antagonise the binding of [3H]-8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX, ( 9) to the A1 adenosine receptor of DDT1 MF-2 cells. For comparison, the xanthines were also tested for their ability to inhibit the binding of [3H]-4-(2-[7-amino-2-{furyl} {1,2,4}- triazolo{2,3-a} {1,3,5}triazin-5-ylamino-ethyl)]phenol ([3H]ZM241385, 36) to the A2A adenosine receptor of PC-12 cells. The results suggest that the length and chemical composition of the linker separating the reactive 4-(fluorosulfonyl)phenyl moiety from the xanthine ring contribute to the potency and efficacy of the irreversible A1 adenosine receptor ligands. Like FSCPX (44, IC50 A1 = 11.8 nM), all derivatives possessed IC50 values in the low nM range under in vitro conditions. Compounds 94 (IC50 A1 = 165 nM), 95 (IC50 A1 = 112 nM) and 96 (IC50 A1 = 101 nM) possessing one, three and five methylene spacers within the linkage respectively, exhibited potent and selective binding to the A1 adenosine receptor versus the A2A adenosine receptor. Compound 94 did not exhibit any irreversible binding at A1 adenosine receptors, while 95 and 96 exhibit only weak irreversible binding at A1 adenosine receptors. Those compounds containing a benzylic carbonyl separating the 4-(fluorosulfonyl)phenyl moiety from the xanthine ring in the form of an amide (119, IC50 A1 = 24.9 nM, and 120, IC50 A1 = 21 nM) or ketone (151, IC50 A1 = 14 nM) proved to be the most potent, with compound 120 exhibiting the highest selectivity of 132-fold for the A receptor over the A2A receptor. compounds 119, 120 and 151 also strongly inhibited the binding of [3H]DPCPX irreversibly (82%, 83% and 78% loss of [3H]DPCPX binding at 100 nM respectively). compounds 120 and 151 are currently being evaluated for use in in vivo studies. Structure-activity studies suggest that altering the 8-cycloalkyl group of A1 selective xanthines for a 3-substituted or 2,3-disubstituted styryl, combined with N-7 methyl substitution will produce a compound with high affinity and selectivity for the A2A adenosine receptor over the A1 adenosine receptor. Compound 167 (IC50 A2A = 264 nM) possessing 8-(m-chloro)styryl substitution and the reactive 4-(fluorosulfonyl)phenyl moiety separated from the xanthine ring via an amide linker in the 3-position (as for 119 and 120), exhibited relatively potent binding to the A2A adenosine receptor of PC-12 cells, with a 16-fold selectivity for that receptor over the A1 adenosine receptor. However, compound 167 exhibited only very weak irreversible binding at A2A adenosine receptors. Overall, at this stage of biological testing, compound 120 appears to possess the most advantageous characteristics as an irreversible antagonist for the A1 adenosine receptor. This can be attributed to its high selectivity for the A1 adenosine receptor as compared to the A2A adenosine receptor. It also has relatively high potency for the A1 adenosine receptor, a concentration-dependent and selective inactivation of A1 adenosine receptors, and unbound ligand is easily removed (washed out) from biological membranes. These characteristics mean compound 151 has the potential to be a useful tool for the further study of the structure and function of the A1 adenosine receptor.

Study of the physiology and biochemistry of chlorophyllase in Zostera muelleri

Examines the biochemistry and physiology of chlorophyllase in the marine angiosperm Zostera muelleri. Enzyme activity was found to be highly correlated with chlorophyll synthesis in developing leaf tissue. This study provides evidence for the physiological role of chlorophyllase in chlorophyll degradation and provides insights into the senescence process in seagrass.

Docosapentaenoic acid (22:5n-3) : a review of its biological effects

This article summarizes the current knowledge available on metabolism and the biological effects of n-3 docosapentaenoic acid (DPA). n-3 DPA has not been extensively studied because of the limited availability of the pure compound. n-3 DPA is an elongated metabolite of EPA and is an intermediary product between EPA and DHA. The literature on n-3 DPA is limited, however the available data suggests it has beneficial health effects. In vitro n-3 DPA is retro-converted back to EPA, however it does not appear to be readily metabolised to DHA. In vivo studies have shown limited conversion of n-3 DPA to DHA, mainly in liver, but in addition retro-conversion to EPA is evident in a number of tissues. n-3 DPA can be metabolised by lipoxygenase, in platelets, to form ll-hydroxy-7,9,13,16,19- and 14-hydroxy-7,10,12,16,19-DPA. It has also been reported that n-3 DPA is effective (more so than EPA and DHA) in inhibition of aggregation in platelets obtained from rabbit blood. In addition, there is evidence that n-3 DPA possesses 10-fold greater endothelial cell migration ability than EPA, which is important in wound-healing processes. An in vivo study has reported that n-3 DPA reduces the fatty acid synthase and malic enzyme activity levels in n-3 DPA-supplemented mice and these effects were stronger than the EPA-supplemented mice. Another recent in vivo study has reported that n-3 DPA may have a role in attenuating age-related decrease in spatial learning and long-term potentiation. However, more research remains to be done to further investigate the biological effects of this n-3 VLCPUFA.

植物CytP450和抗氧化酶对土壤低浓度菲、芘胁迫的响应

Wheat (Triticum aestivum) was chosen to analyze the effect of two polycyclic aromatic hydrocarbons (PAHs), Phenanthrene (PHE) and Pyrene (PY) in brown meadow soil at low concentrations. The effects of PHE and PY were determined by analyzing the changes in activity of Cytochrome P450 (CytP450) and antioxidant enzymes superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT). Results indicated that both PHE and PY caused changes in activity of CytP450 and the antioxidant enzymes, SOD, POD and CAT. CytP450 activity was significantly stimulated with 1 mg kg-1 of both PHE and PY individually and significantly inhibited with 4 mg kg-1, which showed that pollution stress of PHE or PY can damage the metabolism and detoxification systems of plants. Moreover, as PHE and PY combined at 1 mg kg-1, CytP450 was increased significantly more than when PHE and PY were applied individually, which illustrates obvious synergistic effects. No significant variation were found in activity of SOD in response to individual exposure of PHE or PY in soil, but SOD activity decreased slightly in response to a combined PHE and PY exposure. Great decrease variation was found in CAT and POD activity in response to individual exposure of PHE or PY in soil. No enhanced toxic effects were shown by POD in response to a PHE and PY combined exposure, however CAT showed increased inhibition. From the aspects of metabolism and detoxification as well as antioxidant enzyme activity, our study has provided experimental basis for the pollution diagnosis of PAHs in soils at low concentrations.