Vivian Hsiu-Chuan Liao

Arsenite-oxidizing and arsenate-reducing bacteria associated with arsenic-rich groundwater in Taiwan

Drinking highly arsenic-contaminated groundwater is a likely cause of blackfoot disease in Taiwan, but microorganisms that potentially control arsenic mobility in the subsurface remain unstudied. The objective of this study was to investigate the relevant arsenite-oxidizing and arsenate-reducing microbial community that exists in highly arsenic-contaminated groundwater in Taiwan. We cultured and identified arsenic-transforming bacteria, analyzed arsenic resistance and transformation, and determined the presence of genetic markers for arsenic transformation. In total, 11 arsenic-transforming bacterial strains with different colony morphologies and varying arsenic transformation abilities were isolated, including 10 facultative anaerobic arsenate-reducing bacteria and one strictly aerobic arsenite-oxidizing bacterium. All of the isolates exhibited high levels of arsenic resistance with minimum inhibitory concentrations of arsenic ranging from 2 to 200 mM. Strain AR-11 was able to rapidly oxidize arsenite to arsenate at concentrations relevant to environmental groundwater samples without the addition of any electron donors or acceptors. We provide evidence that arsenic-reduction activity may be conferred by the ars operon(s) that were not amplified by the designed primers currently in use. The 16S rRNA sequence analysis grouped the isolates into the following genera: Pseudomonas, Bacillus, Psychrobacter, Vibrio, Citrobacter, Enterobacter, and Bosea. Among these genera, we present the first report of the genus Psychrobacter being involved in arsenic reduction. Our results further support the hypothesis that bacteria capable of either oxidizing arsenite or reducing arsenate coexist and are ubiquitous in arsenic-contaminated groundwater.

Cadmium-regulated genes from the nematode Caenorhabditis elegans. Identification and cloning of new cadmium-responsive genes by differential display.

The transition metal cadmium is a pervasive and persistent environmental contaminant that has been shown to be both a human toxicant and carcinogen. To inhibit cadmium-induced damage, cells respond by increasing the expression of genes encoding stress-response proteins. In most cases, the mechanism by which cadmium affects the expression of these genes remains unknown. It has been demonstrated in several instances that cadmium activates gene transcription through signal transduction pathways, mediated by protein kinase C, cAMP-dependent protein kinase, or calmodulin. A codicil is that cadmium should influence the expression of numerous genes. To investigate the ability of cadmium to affect gene transcription, the differential display technique was used to analyze gene expression in the nematode Caenorhabditis elegans. Forty-nine cDNAs whose steady-state levels of expression change 2–6-fold in response to cadmium exposure were identified. The nucleotide sequences of the majority of the differentially expressed cDNAs are identical to those of C. elegans cosmids, yeast artificial chromosomes, expressed sequence tags, or predicted genes. The translated amino acid sequences of several clones are identical to C. elegansmetallothionein-1, HSP70, collagens, and rRNAs. In addition, C. elegans homologues of pyruvate carboxylase, DNA gyrase, β-adrenergic receptor kinase, and human hypothetical protein KIAA0174 were identified. The translated amino acid sequences of the remaining differentially expressed cDNAs encode novel proteins.

Curcumin-mediated lifespan extension in Caenorhabditis elegans

Curcumin is the active ingredient in the herbal medicine and dietary spice, turmeric (Curcuma longa). It has a wide range of biological activities, including anti-inflammatory, antioxidant, chemopreventive, and chemotherapeutic activities. We examined the effects of curcumin on the lifespan and aging in Caenorhabditis elegans, and found that it responded to curcumin with an increased lifespan and reduced intracellular reactive oxygen species and lipofuscin during aging. We analyzed factors that might influence lifespan extension by curcumin. We showed that lifespan extension by curcumin in C. elegans is attributed to its antioxidative properties but not its antimicrobial properties. Moreover, we showed that lifespan extension had effects on body size and the pharyngeal pumping rate but not on reproduction. Finally, lifespan tests with selected stress- and lifespan-relevant mutant strains revealed that the lifespan-extending phenotype was absent from the osr-1, sek-1, mek-1, skn-1, unc-43, sir-2.1, and age-1 mutants, whereas curcumin treatment prolonged the lifespan of mev-1 and daf-16 mutants. Our study has unraveled a diversity of modes of action and signaling pathways to longevity and aging with curcumin exposure in vivo.

Molecular Characterization of a Novel, Cadmium-inducible Gene from the Nematode Caenorhabditis elegans A NEW GENE THAT CONTRIBUTES TO THE RESISTANCE TO CADMIUM TOXICITY

Cadmium is an environmental contaminant that is both a human toxicant and carcinogen. To inhibit cadmium-induced damage, cells respond by increasing the expression of genes that encode stress-response proteins. We previously reported the identification of 48 cadmium-inducible mRNAs in the nematode Caenorhabditis elegans. Here we describe a new cadmium-responsive gene, designated cdr-1, whose rate and level of inducible expression parallel those of the C. elegansmetallothioneins. The CDR-1 mRNA contains an open reading frame of 831 bp and encodes a predicted 32-kDa, integral membrane protein. Following cadmium exposure, cdr-1 is transcribed exclusively in intestinal cells of post-embryonic C. elegans. In vivo, the CDR-1 protein is targeted specifically to the intestinal cell lysosomes. cdr-1transcription is significantly induced by cadmium but not by other tested stressors. These results indicate that cdr-1expression is regulated by cadmium and in a cell-specific fashion. Inhibition of CDR-1 expression renders C. eleganssusceptible to cadmium toxicity. In conclusion, cdr-1defines a new class of cadmium-inducible genes and encodes an integral membrane, lysosomal protein. This protein functions to protect against cadmium toxicity.

Lung cancer risk in relation to traffic-related nano/ultrafine particle-bound PAHs exposure: A preliminary probabilistic assessment

Exposures to carcinogenic polycyclic aromatic hydrocarbons (PAHs) have been linked to human lung cancer. The purpose of this study was to assess lung cancer risk caused by inhalation exposure to nano/ultrafine particle-bound PAHs at the population level in Taiwan appraised with recent published data. A human respiratory tract model was linked with a physiologically based pharmacokinetic model to estimate deposition fraction and internal organic-specific PAHs doses. A probabilistic risk assessment framework was developed to estimate potential lung cancer risk. We reanalyzed particle size distribution, total-PAHs, particle-bound benzo(a)pyrene (B[a]P) and PM concentrations. A dose-response profile describing the relationships between external B[a]P concentration and lung cancer risk response was constructed based on population attributable fraction (PAF). We found that 90% probability lung cancer risks ranged from 10(-5) to 10(-4) for traffic-related nano and ultrafine particle-bound PAHs, indicating a potential lung cancer risk. The particle size-specific PAF-based excess annual lung cancer incidence rate due to PAHs exposure was estimated to be less than 1 per 100,000 population, indicating a mild risk factor for lung cancer. We concluded that probabilistic risk assessment linked PAF for limiting cumulative PAHs emissions to reduce lung cancer risk plays a prominent role in future government risk assessment program.

Development and testing of a green fluorescent protein-based bacterial biosensor for measuring bioavailable arsenic in contaminated groundwater samples

A green fluorescent protein (GFP)-based bacterial biosensor for the detection of bioavailable As(III), As(V), and Sb(III) was developed and characterized. The biosensor strain Escherichia coli DH5alpha (pVLAS1) was developed based on the expression of gfp under the control of the ars promoter and the arsR gene of Staphylococcus aureus plasmid pI258. Strain DH5alpha (pVLAS1) responded mainly to As(III), As(V), and Sb(III), with the lowest detectable concentrations being 0.4, 1, and 0.75 microM, respectively, during a 2-h exposure and 0.1 microM for all three metal ions with an 8-h induction period. To assess its applicability for analyzing environmentally relevant samples, the biosensor was field-tested on shallow-well groundwater for which contaminant levels were known. Our results demonstrate that the nonpathogenic bacterial biosensor developed in the present study is useful and applicable in determining the bioavailability of arsenic with high sensitivity in contaminated groundwater samples, and they suggest a potential for its inexpensive application in field-ready tests.

Monascin from red mold dioscorea as a novel antidiabetic and antioxidative stress agent in rats and Caenorhabditis elegans

Monascin is a major yellow compound from red mold dioscorea. We investigated monascin to test whether this compound acts as an antidiabetic and antioxidative stress agent in diabetic rats and Caenorhabditis elegans. The mechanisms by which monascin exerts its action in vivo were also examined. Streptozotocin (STZ)-induced diabetic rats were given monascin at 30 mg/kg/day and sacrificed after 8 weeks. Blood glucose and serum insulin, triglyceride, total cholesterol, and high-density lipoprotein and antioxidative enzymes in the pancreas of rats were measured. In addition, monascin was evaluated for stress resistance and potential associated mechanisms in C. elegans. Throughout the 8-week experimental period, significantly lowered blood glucose, serum triglyceride, and total cholesterol and higher high-density lipoprotein levels were observed in monascin-treated rats. Monascin-treated rats showed higher serum insulin level, lower reactive oxygen species production, and higher activities of glutathione peroxidase, superoxide dismutase, and catalase in the pancreas compared to diabetic control rats. In addition, monascin significantly induced the hepatic mRNA levels of FOXO3a, FOXO1, MnSOD, and catalase in STZ-induced diabetic rats. Monascin-treated C. elegans showed an increased survival rate during oxidative stress and heat stress treatments compared to untreated controls. Moreover, monascin extended the life span under high-glucose conditions and enhanced expression of small heat shock protein (sHSP-16.2), superoxide dismutase (SOD-3), and glutathione S-transferase (GST-4) in C. elegans. Finally, we showed that monascin affected the subcellular distribution of the FOXO transcription factor DAF-16, whereas it was unable to enhance oxidative stress resistance in the daf-16 deletion mutant in C. elegans. Mechanistic studies in rats and C. elegans suggest that the protective effects of monascin are mediated via regulation of the FOXO/DAF-16-dependent insulin signaling pathway by inducing the expression of stress response/antioxidant genes, thereby enhancing oxidative stress resistance.

Caenorhabditis elegans gcs-1 confers resistance to arsenic-induced oxidative stress

Gamma-glutamylcysteine synthetase (γ-GCS) catalyzes the first, rate-limiting step in the biosynthesis of glutathione (GSH). To evaluate the protective role of cellular GSH against arsenic-induced oxidative stress in Caenorhabditis elegans (C. elegans), we examined the effect of the C. elegans ortholog of GCS(h), gcs-1, in response to inorganic arsenic exposure. We have evaluated the responses of wild-type and gcs-1 mutant nematodes to both inorganic arsenite (As(III)) and arsenate (As(V)) ions and found that gcs-1 mutant nematodes are more sensitive to arsenic toxicity than that of wild-type animals. The amount of metal ion required to kill half of the population of worms falls in the order of wild-type/As(V)>gcs-1/As(V)> wild-type/As(III)>gcs-1/As(III). gcs-1 mutant nematodes also showed an earlier response to the exposure of As(III) and As(V) than that of wild-type animals. Pretreatment with GSH significantly raised the survival rate of gcs-1 mutant worms compared to As(III)- or As(V)-treated worms alone. These results indicate that GCS-1 is essential for the synthesis of intracellular GSH in C. elegans and consequently that the intracellular GSH status plays a critical role in protection of C. elegans from arsenic-induced oxidative stress.

In Vivo Antioxidant Activities of Essential Oils and Their Constituents from Leaves of the Taiwanese Cinnamomum osmophloeum

Cinnamomum osmophloeum Kaneh is an indigenous tree species in Taiwan. In this study, phytochemical characteristics and antioxidant activities of the essential oils and key constituents from the leaves of two C. osmophloeum clones were investigated. The two trees possess two chemotypes, which were classified as the cinnamaldehyde type and camphor type. We demonstrated that the essential oils from C. osmophloeum leaves exerted in vivo antioxidant activities in Caenorhabditis elegans. In addition, trans-cinnamaldehyde and D-(+)-camphor, which respectively represent the major compounds in the cinnamaldehyde-type and camphor-type trees, exerted significant in vivo antioxidant activities against juglone-induced oxidative stress in C. elegans. Moreover, expressions of antioxidative-related genes, including superoxide dismutase (SOD) and glutathione S-transferase (GST), were significantly induced by trans-cinnamaldehyde and D-(+)-camphor from C. osmophloeum leaves. Our results showed that the essential oils from C. osmophloeum leaves and their major compounds might have good potential for further development as nutraceuticals or antioxidant remedies.

Acute toxicity and bioaccumulation of arsenic in freshwater clam Corbicula fluminea

Arsenic is a potent human carcinogen of skin, lung, and urinary bladder. Freshwater clam Corbicula fluminea is a commercially important native species in Taiwan. C. fluminea is also a suitable biomonitoring test organism. Little is known, however, about the actual effects of arsenic on C. fluminea. The objectives of this study were to provide information on the acute toxicity and bioaccumulation kinetics of arsenic in C. fluminea. We carried out a 14‐day exposure experiment to obtain bioaccumulation parameters. Uptake was very rapid when C. fluminea was first exposed and then slightly decayed during the uptake phase of the experiment and an uptake rate constant of 1.718 ± 6.70 (mean ± SE) mL g−1 d−1 was estimated. The elimination of arsenic from C. fluminea obeyed first‐order depuration kinetics (r2 = 0.85, p < 0.05) with a calculated half‐life of 6.80 days. The derived bioaccumulation factor of 16.84 suggests that arsenic has a high potential for bioaccumulation in C. fluminea. This had important implications for dietary exposure of arsenic to humans who eat contaminated clams, because the soft tissue usually constitutes the majority of tissue consumed. The 96‐h LC50 value was estimated to be 20.74 (95% CI: 11.74–30.79) mg L−1 obtained from a 7‐day acute toxicity bioassay. We also kinetically linked an acute toxicity model and a Hill sigmoid model to reconstruct an internal effect concentration based dose‐response profile to assess the effect of soft tissue arsenic burden on the C. fluminea mortality. This result could be used to support the establishment of an ecological risk assessment to prevent possible ecosystem and human health consequences.