Genotoxicity assessment of soils from wastewater irrigation areas and bioremediation sites using the Vicia faba root tip micronucleus assay

Genotoxicity potential of soils taken from wastewater irrigation areas and bioremediation sites was assessed using the Vicia faba root tip micronucleus assay. Twenty five soils were tested, of which 8 were uncontaminated soils and taken as the control to examine the influence of soil properties; 6 soils were obtained from paddy rice fields with a history of long-term wastewater irrigation; 6 soils were obtained from bioremediation sites to examine effects of bioremediation; and 5 PAH-contaminated soils were used to examine methodological effects between direct soil exposure and exposure to aqueous soil extracts on micronuclei (MN) frequency () in the V. faba root tips. Results indicate that soil properties had no significant influences on MN frequencies (p > 0.05) when soil pH varied between 3.4 to 7.6 and organic carbon between 0.4% and 18.6%. The MN frequency measured in these control soils ranged from 1.6‰ to 5.8‰. MN frequencies in soils from wastewater irrigation areas showed 2- to 48-fold increase as compared with the control. Soils from bioremediation sites showed a mixed picture: MN frequencies in some soils decreased after bioremediation, possibly due to detoxification; whereas in other cases remediated soils induced higher MN frequencies, suggesting that genotoxic substances might be produced during bioremediation. Exposure to aqueous soil extracts gave a higher MN frequency than direct exposure in 3 soils. However, the opposite was observed in the other two soils, suggesting that both exposure routes should be tested in case of negative results from one route. Data obtained from this study indicate that the MN assay is a sensitive assay suitable for evaluating genotoxicity of soils.

Examination of the role of intestinal Fatty acid-binding protein in drug absorption using a parallel artificial membrane permeability assay

Transcellular diffusion across the absorptive epithelial cells (enterocytes) of the small intestine is the main route of absorption for most orally administered drugs. The process by which lipophilic compounds transverse the aqueous environment of the cytoplasm, however, remains poorly defined. In the present study, we have identified a structurally diverse group of lipophilic drugs that display low micromolar binding affinities for a cytosolic lipid-binding protein—intestinal fatty acid-binding protein (I-FABP). Binding to I-FABP significantly enhanced the transport of lipophilic drug molecules across a model membrane, and the degree of transport enhancement was related to both drug lipophilicity and I-FABP binding affinity. These data suggest that intracellular lipid-binding proteins such as I-FABP may enhance the membrane transport of lipophilic xenobiotics and facilitate drug access to the enterocyte cytoplasm and cytoplasmic organelles.

Studies on dietary fibre : analysis, epidemiological and physiological aspects

This thesis involves an investigation in three areas; first, a study of an enzymatic-gravimetric method for the analysis of dietary fibre; second, a survey of dietary fibre intake in an area of a developing country, and finally, some observations on the functional aspects of gel-forming dietary fibre in the rat. A simple and rapid enzymatic-gravimetric assay for both soluble and insoluble dietary fibre has been critically investigated. Reference samples were also analysed by a more comprehensive, enzymatic gas chromatographic method to allow testing of the relative accuracy of the enzymatic-gravimetric method. The enzymatic-gravimetric method was found to be highly reproducible but gave a slightly higher value for total dietary fibre than the more comprehensive method. This discrepancy is probably due to the presence of small quantities of resistant starch and protein residue which are recovered in the enzymatic-gravimetric method. In the enzymatic-gas chromatographic method, protein residue is not measured, and resistant starch is estimated, but not counted as dietary fibre. The enzymatic-gravimetric method was applied to the analysis of foods commonly consumed in the Padang region of West Sumatra in Indonesia, in order to estimate dietary fibre intake in the region. Daily intakes of usual foods were estimated by use of a 24-hour recall procedure aided by food photographs to assist in the estimation of portion size. Samples of approximately 60 of the most commonly consumed foods were collected and analysed for dietary fibre. These appear to be the first data which report values for dietary fibre in Indonesion foods and they represent a significant improvement upon the existing data on crude fibre content. Knowledge of the amounts of foods usually consumed and their dietary fibre content allowed an estimation of usual intakes of dietary fibre. Fibre intake was found to be lower than in the developing countries of Africa and was comparable to intakes measured in the U.K. This is the first study to show that in this part of South East Asia, a developing country area using polished rice as a staple food, dietary fibre intakes are as low as in Western countries. Low intakes of fibre are believed to be related to the prevalence of a range of diseases and, in this study, preliminary data on the rates of non-infective, chronic diseases were collected from the two main hospitals in West Sumatra. Chronic, non-infectious diseases such as inguinal hernia, appendicitis, haemorrhoids, diabetes mellitus, hypertension and malignant neoplasms of the rectum are relatively frequent in West Sumatra. While no firm conclusions can be drawn from these data, they do show the possibility of a relationship between low intakes of dietary fibre and the prevalence of these diseases, and suggest that further investigation is necessary. Some observations were made of the effect of gel-forming dietary fibre on stomach emptying and intestinal transit rate in the rat. Xanthan gum was added to iso-osmotic solutions to produce increased viscosity and phenol sulphonphthalein (phenol red) was used as a non-absorbable marker. Gavage feeding of solutions with a range of viscosities was used to study the effect of viscosity on the rate of stomach emptying and intestinal transit. Increased viscosity was observed to slow gastro-intestinal transit and this provides one mechanism by which dietary fibre of the gel-forming type ray improve glucose tolerance.

Utilization of a new bdelloid rotifer (Philodina acuticornis odiosa) assay to evaluate the effect of salinity on the toxicity of chlorothalonil

Acute (24 h) toxicity tests were conducted to determine the toxicity of the fungicide chlorothalonil towards the freshwater bdelloid rotifer (Philodina acuticornis odiosa). Since rotifers are the dominant zooplankton species in many inland freshwater lakes in Australia, the influence of salinity on chlorothalonil toxicty was also assessed. The rotifers used in this study appeared to be reasonably tolerant to changes in salinity, with little mortality observed at 3760 µS cm-1, increasing thereafter at higher salinity. The bdelloid rotifers were, however, found to be highly sensitive to chlorothalonil (24 h LC50, 3.2 µg L-1) with results also suggesting that as salinity increases, so does toxicity (e.g., 24 h LC50 at 5000 µS cm-1, 0.5 µg L-1).

High-performance liquid chromatography with post-column 2,2′-diphenyl-1-picrylhydrazyl radical scavenging assay : methodological considerations and application to complex samples

An improved post-column 2,2´-diphenyl-1-picrylhydrazyl (DPPH•) radical scavenging assay for the screening of antioxidants in complex matrices was developed. Experimental parameters believed to be influential to DPPH• response were studied in a univariate approach. Optimum conditions were found to be: 5×10⁻⁵M DPPH• reagent prepared in a 75% methanol: 25% 40mM citric acid–sodium citrate buffer (pH 6) solution, degassed with nitrogen; reaction coil of 2m×0.25mm i.d. PEEK tubing; detection at 521 nm; analysis at room temperature. The analytical utility of this protocol was evaluated by screening for antioxidants in thyme and green tea, in comparison with two commonly employed methodologies.

Correlation between acidic potassium permanganate chemiluminescence and in vitro cell culture assay : physiologically meaningful antioxidant activity

There is great interest in the activity of antioxidant molecules, including polyphenols, from food and plant sources. Acidic potassium permanganate chemiluminescence signal intensity was shown to predict the ability of polyphenols to positively act on cellular redox state and attenuate oxidative stress in cultured skeletal muscle cells.

Molecular characterization and enzymatic hydrolysis of flavanoid extracted from citrus waste

The citrus fruit processing industry generates substantial quantities of waste rich in phenolic substances, which is a valuable natural source of polyphenols (flavonoids) such as naringin and its disposal is becoming a major problem. In the US alone, the juice processing of oranges and grapefruit generates over 5 Mt of citrus waste every year. In the case of India, about 2.15 Mt of citrus peel out of 6.28 Mt of citrus fruits are produced yearly from citrus juice processing. In case of Australia, about 15-40% of citrus peel waste is generated by processing of citrus fruit (0.85 Mt). Thus Isolation of functional compounds (mostly flavanoids) and their further processing can be of interest to the food and pharmaceutical industry. This peel is rich in naringin and may be used for rhamnose production by utilizing α-L-rhamnosidase (EC 3.2.1.40), an enzyme that catalyzes the cleavage of terminal rhamnosyl groups from naringin to yield prunin and rhamnose. We recently purified recombinant α-L-rhamnosidase from E. coli cells using immobilized metal-chelate affinity chromatography (IMAC) and used it for naringin hydrolysis. The purified enzyme established hydrolysis of naringin extracted from citrus peel and thus endorses its industrial applicability for producing rhamnose. Infrared (IR) spectroscopy confirmed molecular characteristics of naringin extracted from citrus peel waste.

Role of glutathione in schizophrenia? The effect of brain glutathione depletion on prepulse inhibition (PPI) in rats and mice

Reductions in brain glutathione (GSH) levels have been reported in schizophrenia. We investigated the effects of brain GSH depletion on prepulse inhibition (PPI), a model of sensorimotor gating which is disrupted in individuals with schizophrenia. It was hypothesized that GSH depletion would lead to disruption of PPI similar to that seen in schizophrenia and enhance the effect of increased dopamine release by amphetamine. Sprague-Dawley rats and C57Bl/6 mice were treated with saline or 2-cyclohexene-1-one (CHX, 75 mg/kg and 120 mg/kg respectively) to deplete brain GSH. 225 minutes later the animals were injected with amphetamine (2.5 mg/kg in rats and 25 mg/kg in mice). Total brain GSH levels were measured using an enzymatic recycling assay. Surprisingly, in rats CHX treatment prevented the disruption of PPI by amphetamine. Thus, while there was the expected disruption of PPI caused by amphetamine on its own (average %PPI reduced from 58 ± 5 to 44 ± 4), in combination with CHX, amphetamine had no significant effect (67 ± 4 vs. 63 ± 3, respectively). In contrast to rats, in mice CHX had no effect on PPI. Thus, amphetamine similarly disrupted PPI after saline (41 ± 5 vs. 28 ± 5) and CHX pretreatment (45 ± 6 vs. 26 ± 5). There were significant 40-63% depletions of GSH in frontal cortex and striatum of CHX-treated rats and mice. These data show that GSH depletion in the brain by CHX treatment did not induce the expected decrease in PPI. Because the levels of GSH depletion in this study were similar to those found in schizophrenia, these results cast doubt on a direct interaction between brain GSH levels and PPI disruption in this illness. In rats, CHX treatment prevented the disruption of PPI caused by amphetamine. We have observed that resting levels of GSH are lower in rats than in mice. It is plausible that some oxidative damage may occur after amphetamine treatment alone, which induces marked release of the electroactive species, dopamine. In mice with their higher levels of GSH (either with or without CHX treatment) and in control rats, this does not cause functional effects. However, in CHX-treated rats GSH levels are reduced to a point where amphetamine-induced dopamine release may cause increased metabolism and lipid peroxidation inducing a decrease in postsynaptic dopamine receptor function and consequently leading to an apparent inhibition of the disruption of PPI. In conclusion, while individuals with schizophrenia show disruption of PPI and reduced brain GSH levels, in rats and mice brain GSH depletion alone does not impact on PPI. In combination with a hyperdopaminergic state, functional effects on PPI regulation were found. These effects warrant further investigation.

A gel-capture assay for characterizing the sialyl-glycan selectivity of influenza viruses

Sialic acids (SA) usually linked to galactose (Gal) in an α2,6- or α2,3-configuration are considered the main cell receptors for influenza viruses, in particular for their hemagglutinins (HA). The typing of influenza virus HA receptor selectivity is relevant for understanding the transmissibility of avian and swine viruses to the human population. In this study we developed a simple and inexpensive gel-capture assay (GCA) of the influenza virus HA receptor-binding selectivity. Its principle is the binding of soluble influenza virus to pentasaccharide analogs, representatives of receptors of human and avian influenza viruses, immobilized on a gel resin. The human and avian analogs consisted of a sialyllactose-N-tetraose c (LSTc) [Neu5Ac(α2,6)Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glc] and a sialyllactose-N-tetraose a (LSTa) [Neu5Ac(α2,3)Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glc], respectively. Following equilibration, the unbound virus is washed away and the bound one is assayed via HA by densitometry as a function of the analog concentration. Using GCA, the receptor selectivity of three influenza viruses of different HA subtype was investigated. The results showed that the egg-adapted A/California/07/2009 (H1N1) virus exhibited an avian α2,3-linked LSTa selectivity, however, it retained the ability to bind to the α2,6-linked LSTc human receptor analog. Influenza B virus B/Florida/4/2006 showed α2,6-linked LSTc selectivity and a poor α2,3-linked LSTa avidity. The H3N2 virus A/Wisconsin/15/2009 displayed almost comparable avidity for both receptor analogs with a marginally greater α2,3-linked LSTa avidity. The described assay protocol provides a simple and rapid method for the characterization of influenza virus HA receptor binding selectivity. Keywords: influenza virus; hemagglutinin; receptor; sialyllactose-N-tetraose; gel-capture assay.