Sublethal Concentrations Of Triclosan Research Articles

Effects of acute triclosan exposure on gill and liver tissues of zebrafish (Danio rerio)

Triclosan (TCS) is a broad-spectrum antimicrobial agent found in personal care, household, veterinary, medical, and industrial products. TCS has been detected in soil, sediment, and surface water. In the current work, the effects of acute exposure to TCS on gill and liver tissues of zebrafish were evaluated. Adult zebrafish were treated with sublethal concentrations of TCS (34, 85, and 170 μg/L) for 120 h. Gill and liver samples were removed and embedded in paraffin for histopathological investigations. TCS induced aneurysm, capillary dilation, lamellar disorganization, hyperplasia, epithelial lifting, and desquamation in the gills. Liver specimens showed sinusoidal dilation, congestion, vacuolization, hepatocellular degeneration, and necrosis. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed to observe the apoptotic effect of TCS. Accordingly, no apoptotic cells were detected in the specimens of 34 μg/L of TCS exposed gills, while the slides of 85 and 170 μg/L of TCS treated gills showed a high number of TUNEL-positive cells. In the liver, no TUNEL-positive cells were identified in 34 μg/L of TCS treated samples of zebrafish. 85 and 170 μg/L of TCS exposed livers showed few hepatic parenchymal cells undergoing apoptosis.

Histopathological and apoptotic examination of zebrafish (Danio rerio) gonads exposed to triclosan

Triclosan, produced as a broad-spectrum antibiotic in the early 1960s, is generally used as a preservative in personal care products, fabrics, plastic products such as kitchenware and toys. As a result of the high demand for triclosan, this chemical threatens the aquatic ecosystem by contaminating wastewater sources. Environmental pollutants affect the reproductive potential of fish, one of the most critical aquatic organisms. This study aimed to investigate the histopathological and apoptotic effects of triclosan in zebrafish gonads. Fish were exposed to sublethal concentrations of triclosan for 5 days, and general histological methods were applied. Histological sections were examined under a light microscope after staining with hematoxylin and eosin and toluidine blue. Triclosan exposure caused deterioration in ovarian tissue, such as shrinkage in the ooplasm, accumulation of proteinaceous fluid in the interstitial tissue, morphological changes of oocyte and the zona radiata. In testicular tissue, triclosan exposure caused fusion in seminiferous tubules, hypertrophy in spermatogenic and Leydig cells, edema in seminiferous tubules, and karyorrhexis in spermatogenic cells. The TUNEL assay was used for the determination of apoptotic cells. Brown-colored apoptotic cells were visualized under the light microscope. TUNEL positive cells were observed in all exposure groups. Triclosan administration was found to cause apoptosis in zebrafish gonads. These findings indicate that triclosan potentially affects fish reproduction, and that its judicious disposal is essential for protecting the environment and maintaining the reproductive potential of fish.

Triclosan affects motor function in zebrafish larva by inhibiting ache and syn2a genes

The widespread use of triclosan in personal care products as an antimicrobial agent is leading to its alarming tissue-bioaccumulation including human brain. However, knowledge of its potential effects on the vertebrate nervous system is still limited. Here, we hypothesized that sublethal triclosan concentrations are potent enough to alter motor neuron structure and function in zebrafish embryos exposed for prolonged duration. In this study, zebrafish embryos were used as vertebrate-animal model. Prolonged exposure (up to 4 days) of 0.6 mg/L (LC50, 96 h) and 0.3 mg/L (<LC50, Sublethal) triclosan produced aberrations in motor neuron innervations in skeletal muscles and reduced touch-evoked escape response in zebrafish larvae. This suggests motor dysfunction in treated embryos. To further explore the mechanisms of triclosan induced neurotoxicity, we determined the enzyme activity of acetylcholinesterase (AChE) and the expression of acetylcholinesterase (ache), myelin basic protein (mbp) and synapsin IIa (syn2a) genes which play an important role in the neural development and synaptic transmission. The ache and syn2a genes were down-regulated in triclosan treated larvae without any significant changes in mbp gene expression. At functional level, we observed a decrease in the AChE activity. Furthermore, docking results showed that triclosan can form a stable interaction with binding pocket of AChE and perhaps it can compete with natural acetylcholine for direct binding to AChE thereby inhibiting it and affecting cholinergic transmission. Therefore, triclosan can be regarded as a neurotoxic agent even at sublethal concentrations. Overall, the growing toxicological evidence against triclosan including ours suggest caution in its widespread use.

Biomarkers for the toxicity of sublethal concentrations of triclosan to the early life stages of carps

Accumulation, contents of protein, non-enzymatic antioxidant glutathione (GSH and GSSG), lipid peroxidation product (melondialdehyde-MDA) and organic acids (fumarate, succinate, malate and citrate), and activities of neurological (acetylcholinesterase-AChE), detoxification (glutathione S-transferase-GST) and metabolic (lactate dehydrogenase-LDH, aspartate transaminase-AST and alanine transaminase-ALT) enzymes were recorded in the hatchlings of Cyprinus carpio, Ctenopharyngodon idella, Labeo rohita and Cirrhinus mrigala after 7 and 14 days exposure and 10 days post exposure (recovery period) to sublethal concentrations (0.005, 0.01, 0.02 and 0.05 mg/L) of triclosan, a highly toxic and persistent biocide used in personal care products. Accumulation was maximum between 7–14 days at 0.01 mg/L for C. carpio and L. rohita but at 0.005 mg/L for C. idella and C. mrigala. No triclosan was observed at 0.005 mg/L in C. carpio and C. mrigala after recovery. Significant decline in protein, glutathione and acetylcholinesterase but increase in glutathione S-transferase, lactate dehydrogenase, aspartate transaminase, alanine transaminase, melondialdehyde and organic acids over control during exposure continued till the end of recovery period. Integrated biomarker response (IBR) analysis depicted higher star plot area for glutathione and glutathione S-transferase during initial 7 days of exposure, thereafter, during 7–14 days of exposure and the recovery period, higher star plot area was observed for acetylcholinesterase, aspartate transaminase, alanine transaminase and organic acids. Higher star plot area was observed for protein in all the species throughout the study. The study shows that L. rohita is most sensitive and glutathione, acetylcholinesterase, aspartate transaminase and alanine transaminase are the biomarkers for the toxicity of sublethal concentrations of TCS.

Sublethal concentrations of triclosan elicited oxidative stress, DNA damage, and histological alterations in the liver and brain of adult zebrafish.

Triclosan (TCS), an antimicrobial agent, has been a pollutant of increasing concern owing to its potential health risk on humans and aquatic animals. The present study seeks to test the hypothesis that TCS could alter the oxidative stress-related parameters in the brain and liver, as well as eliciting DNA damage in hepatocytes of adult zebrafish. On the basis of the 96h LC50 (398.9μg/L), adult zebrafish were separately exposed to 50, 100, and 150μg/L TCS for 30days. The brain and liver tissues from adult zebrafish were excised and assayed for a suite of antioxidant parameters and oxidative stress biomarkers including DNA damage in the liver. The induced effect by TCS on the activity of acetylcholinesterase (AChE) was also analyzed in the brain. Results showed a significant decrease in superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) in the brain and liver of adult zebrafish. Also, the contents of the glutathione system (GSH and GSSH), as well as the activity of the glutathione reductase (GR), assayed in the liver, were reduced while the contents of malondialdehyde (MDA) were elevated in the liver. A comet assay revealed dose-dependent DNA damage in zebrafish hepatocytes. The 8-hydroxy-2'-deoxyguanosine (8-OHdG), MDA, and carbonyl protein contents in brain tissues significantly increased. Moreover, the AChE in the zebrafish brain was induced. Apparently, no obvious histological changes in brain tissues of zebrafish were observed compared with those of the control whereas atrophy and necrosis of hepatocytes and increased hepatic plate gap were observed in zebrafish hepatocytes after TCS exposure. The obtained results highlight that sublethal concentrations of TCS may be deleterious to the liver and brain of adult zebrafish upon subchronic exposure.

A multi-biomarker approach using integrated biomarker response to assess the effect of pH on triclosan toxicity in Pangasianodon hypophthalmus (Sauvage, 1878)

Application of biomarkers is an effective approach for a better understanding of varying toxicity in aquatic organisms during the seasonal and diurnal changes in the natural environment. This report describes the toxicity of sub-lethal concentrations of triclosan (TCS) at different pH (6.5, 7.5 and 8.5) based on selected biomarkers related to oxidative stress, metabolism and genotoxicity in Pangasianodon hypophthalmus. The 96 h LC50 of TCS for P. hypophthalmus was lower at pH 6.5 when compared to higher pH. The sub-lethal concentration of TCS exhibited a significant decrease in hematological parameters related to complete blood counts except for total leukocyte count (TLC), mean cell haemoglobin concentration (MCHC) and red cell distribution width (RDW). Multivariate data analysis showed a significant interaction of TCS and pH in metabolizing enzymes like glutamic oxaloacetate transaminase (GOT), glutamic pyruvic transaminase (GPT), Lactate dehydrogenase (LDH), antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione-s-transferase (GST) and neurotransmitter enzyme acetylcholinesterase (AChE). A significant increase in DNA damage and micronuclei frequency in liver and blood cells of TCS exposed fish at pH 6.5 indicate that the TCS exposure has pronounced effects on genetic materials. The findings of present study establish that enzymes like SOD, LDH, GOT, AChE, DNA damage and micronuclei frequency can be successfully deployed as biomarkers for the assessment of toxicity of TCS in fish.

Impact of triclosan adaptation on membrane properties, efflux and antimicrobial resistance of Escherichia coli clinical isolates.

Analysing the effect of step-wise exposure of Escherichia coli isolates to sublethal concentrations of triclosan (TCS) that is widely used as an antiseptic, preservative and disinfectant. Changes in the tolerance to the biocide itself and the cross-resistance to clinically important antibiotics were analysed. The involvement of efflux mechanism was studied as well as the possible implication of modifications in cytoplasmic membrane properties including integrity, permeability, potential and depolarization in the resistance mechanisms. Most of E.coli isolates that were adapted to TCS showed increased antimicrobial resistance, lower outer and inner membrane permeability, higher membrane depolarization, more negative membrane potential and enhanced efflux activity using qRT-PCR. Nonsignificant change in membrane integrity was found in the adapted cells. This study suggests that the extensive use of TCS at sublethal concentrations contributed to the emergence of antibiotic resistance in E.coli clinical isolates, by inducing changes in bacterial membrane properties and enhancing the efflux system. The extensive usage of TCS has a deleterious effect on the spread of antibiotic resistance, and more studies are needed to investigate the molecular mechanisms underlying the effects of TCS.

Multigenerational effects of triclosan on the demography of Plationus patulus and Brachionus havanaensis (ROTIFERA)

Triclosan is a personal care product widely used in North America, Europe and Asia as antimicrobial ingredient in many consumer chemical products. In Mexico concentrations of triclosan have been reported in aquatic systems. However, there is no law regulating the presence of chemicals such as triclosan, in aquatic systems. The scarce data about this chemical has increased concern among ecotoxicologists regarding possible effects on aquatic organisms. Moreover, multigenerational studies are rarely studied and the results vary depending on the contaminant. Rotifers, are a dominant group of zooplankton, and have been used in aquatic risk assessments of personal care products due to their sensitivity and high reproductive rates. Plationus patulus and Brachionus havanaensis are common rotifers distributed in aquatic ecosystems of Mexico and have been used in ecotoxicological bioassays. In this study, the median lethal concentration (LC50, 24h) of P. patulus and B. havanaensis exposed to triclosan was determined. Based on the LC50, we tested three sublethal concentrations of triclosan to quantify the demographic responses of both rotifers for two successive generations (F0, and F1). The 24h LC50 of triclosan for P. patulus and B. havanaensis were 300 and 500µgL−1 respectively. Despite the concentration, triclosan had an adverse effect on both Plationus patulus and Brachionus havanaensis in both generations exposed. Experiments show that P. patulus was more sensitive than B. havanaensis when exposed to triclosan. When exposed to triclosan the parental generation (F0) of P. patulus was far more affected than F1.

Impacts of triclosan exposure on zebrafish early-life stage: Toxicity and acclimation mechanisms

Triclosan (TCS) is a broad spectrum antibacterial agent widely used in personal care products and present in most aquatic ecosystems. This study investigated the occurrence of triclosan acclimation and the biological mechanisms underlying the stress response triggered in early-life stage of zebrafish. Zebrafish eggs were first exposed to four different sublethal concentrations of TCS (2, 20, 50 and 100μg/L) for 7days following fertilization and subsequently exposed to a lethal concentration of TCS (1000μg/L). During the time-to-death exposure (TTD), mortality was continuously recorded to evaluate if increased resistance occurred. Overall, larvae exposed to 50μg/L of TCS demonstrated higher sensitivity, with delayed hatching and increased mortality during the sub-lethal exposure and significant lower mean time-to-death (TTD) value compared to the other groups. Interestingly, fish exposed to the highest concentration of TCS (100μg/L) presented a similar mean TTD value as controls and a significantly better survival in comparison with embryos exposed to 50μg/L, suggesting that acclimation process has been triggered at this concentration.Proteomic and enzymatic analyses were conducted on 7days post fertilization (dpf) larvae exposed to 50μg/L and 100μg/L of TCS giving insights into the functional changes triggered at those specific concentrations. TCS seemed to affect proteins involved in cytoskeleton, stress response, eyes and neuronal development. This was endorsed by the enzymatic results, which suggest impairment in glutathione metabolism and acute neurotoxicity. A significant 2.5-fold and 3-fold increase of AChE activity was observed following TCS exposure. Moreover, GPx activity was significantly increased whereas a significant inhibition of GR activity was observed, suggesting that de novo synthesis of reduced GSH might occur in order to maintain the ratio between reduced and oxidized GSH. Proteomic results revealed possible candidate protein involved in the acclimation process of larvae exposed to 100μg/L of TCS. Our integrative analysis revealed complex non-monotonic concentration-related effects on zebrafish early-life stages with increased resistance between 50 and 100μg/L exposures. This research highlighted oxidative stress and neurotoxicity as major toxicity mechanisms of TCS during development.

1H NMR-based metabolomics of Daphnia magna responses after sub-lethal exposure to triclosan, carbamazepine and ibuprofen

Pharmaceuticals and personal care products are a class of emerging contaminants that are present in wastewater effluents, surface water, and groundwater around the world. There is a need to determine rapid and reliable bioindicators of exposure and the toxic mode of action of these contaminants to aquatic organisms. 1H nuclear magnetic resonance (NMR)-based metabolomics in combination with multivariate statistical analysis was used to determine the metabolic profile of Daphnia magna after exposure to a range of sub-lethal concentrations of triclosan (6.25–100μg/L), carbamazepine (1.75–14mg/L) and ibuprofen (1.75–14mg/L) for 48h. Sub-lethal triclosan exposure suggested a general oxidative stress condition and the branched-chain amino acids, glutamine, glutamate, and methionine emerged as potential bioindicators. The aromatic amino acids, serine, glycine and alanine are potential bioindicators for sub-lethal carbamazepine exposure that may have altered energy metabolism. The potential bioindicators for sub-lethal ibuprofen exposure are serine, methionine, lysine, arginine and leucine, which showed a concentration-dependent response. The differences in the metabolic changes were related to the dissimilar modes of toxicity of triclosan, carbamazepine and ibuprofen. 1H NMR-based metabolomics gave an improved understanding of how these emerging contaminants impact the keystone species D. magna.

Triclosan-Induced Aminoglycoside-Tolerant Listeria monocytogenes Isolates Can Appear as Small-Colony Variants

Exposure of the human food-borne pathogen Listeria monocytogenes to sublethal concentrations of triclosan can cause resistance to several aminoglycosides. Aminoglycoside-resistant isolates exhibit two colony morphologies: normal-size and pinpoint colonies. The purposes of the present study were to characterize the small colonies of L. monocytogenes and to determine if specific genetic changes could explain the triclosan-induced aminoglycoside resistance in both pinpoint and normal-size isolates. Isolates from the pinpoint colonies grew poorly under aerated conditions, but growth was restored by addition of antibiotics. Pinpoint isolates had decreased hemolytic activity under stagnant conditions and a changed spectrum of carbohydrate utilization compared to the wild type and isolates from normal-size colonies. Genome sequence comparison revealed that all seven pinpoint isolates had a mutation in a heme gene, and addition of heme caused the pinpoint isolates to revert to normal colony size. Triclosan-induced gentamicin-resistant isolates had mutations in several different genes, and it cannot be directly concluded how the different mutations caused gentamicin resistance. However, since many of the mutations affected proteins involved in respiration, it seems likely that the mutations affected the active transport of the antibiotic and thereby caused resistance by decreasing the amount of aminoglycoside that enters the bacterial cell. Our study emphasizes that triclosan likely has more targets than just fabI and that exposure to triclosan can cause resistance to antibiotics that enters the cell via active transport. Further studies are needed to elucidate if L. monocytogenes pinpoint isolates could have any clinical impact, e.g., in persistent infections.

Staphylococcus aureus but not Listeria monocytogenes adapt to triclosan and adaptation correlates with increased fabI expression and agr deficiency

BackgroundThe ability of pathogens to adapt to the widely used biocide, triclosan, varies substantially. The purpose of the study was to examine bacterial adaptation over an extended period of time to low increments of triclosan concentrations. Focus was two human pathogens, S. aureus and L. monocytogenes that previously have displayed inherent high and low adaptability, respectively.ResultsThree strains of L. monocytogenes and two strains of S. aureus including the community-acquired USA300 were exposed to increasing, sub-lethal concentrations of triclosan in triclosan-containing agar gradients. Following 25 days of exposure on agar plates to sub-lethal concentrations of triclosan with a twofold concentration increase every second day, minimum inhibitory concentration (MIC) for S. aureus increased from 0.125 (8325–4) and 0.0625 (USA 300) mg/L to 4 mg/L. The MIC of all three L. monocytogenes strains was initially 4 mg/L and remained unaltered by the exposure. The adapted S. aureus isolates retained normal colony size but displayed increased expression of fabI encoding an essential enzyme in bacterial fatty acid synthesis. Also, they displayed decreased or no expression of the virulence associated agrC of the agr quorum sensing system. While most adapted strains of USA300 carried mutations in fabI, none of the adapted strains of 8325–4 did.ConclusionsAdaptability to triclosan varies substantially between Gram positive human pathogens. S. aureus displayed an intrinsically lower MIC for triclosan compared to L. monocytogenes but was easily adapted leading to the same MIC as L. monocytogenes. Even though all adapted S. aureus strains over-expressed fabI and eliminated expression of the agr quorum sensing system, adaptation in USA300 involved fabI mutations whereas this was not the case for 8325–4. Thus, adaptation to triclosan by S. aureus appears to involve multiple genetic pathways.

Comparative proteomic analysis of Salmonella tolerance to the biocide active agent triclosan

Concern has been expressed about the overuse of biocides in farm animal production and food industries. Biocide application can create selective pressures that lead to increased tolerance to one or more of these compounds and are concomitant with the emergence of cross-resistance to antibiotics. A triclosan sensitive Salmonella enterica serovar Typhimurium and the isogenic triclosan tolerant mutant were studied at the proteomic level in order to elucidate cellular mechanisms that facilitate biocide tolerance. 2-D differential fluorescent gel electrophoresis (DIGE) compared protein profiles of parent and mutant Salmonella, in the presence and absence of triclosan. Differentially expressed proteins were identified by mass spectrometry and divided into two groups: Group A describes proteins differentially expressed between susceptible and triclosan tolerant Salmonella and includes the known triclosan target FabI which contained a mutation at the triclosan target binding site. Group B identified proteins differentially expressed in response to triclosan exposure and defines a general cell defence network. Only four proteins were common to both groups highlighting the diverse range of pathways employed by Salmonella to counteract biocides. These data suggest that sub-lethal concentrations of triclosan induce discernible changes in the proteome of exposed Salmonella and provide insights into mechanisms of response and tolerance.

Attenuated Virulence and Biofilm Formation in Staphylococcus aureus following Sublethal Exposure to Triclosan

Subeffective exposure of Staphylococcus aureus to the biocide triclosan can reportedly induce a small-colony variant (SCV) phenotype. S. aureus SCVs are characterized by low growth rates, reduced pigmentation, and lowered antimicrobial susceptibility. While they may exhibit enhanced intracellular survival, there are conflicting reports regarding their pathogenicity. The current study reports the characteristics of an SCV-like strain of S. aureus created by repeated passage on sublethal triclosan concentrations. S. aureus ATCC 6538 (the passage 0 [P0] strain) was serially exposed 10 times to concentration gradients of triclosan to generate strain P10. This strain was then further passaged 10 times on triclosan-free medium (designated strain ×10). The MICs and minimum bactericidal concentrations of triclosan for P0, P10, and ×10 were determined, and growth rates in biofilm and planktonic cultures were measured. Hemolysin, DNase, and coagulase activities were measured, and virulence was determined using a Galleria mellonella pathogenicity model. Strain P10 exhibited decreased susceptibility to triclosan and characteristics of an SCV phenotype, including a considerably reduced growth rate and the formation of pinpoint colonies. However, this strain also had delayed coagulase production, had impaired hemolysis (P < 0.01), was defective in biofilm formation and DNase activity, and displayed significantly attenuated virulence. Colony size, hemolysis, coagulase activity, and virulence were only partially restored in strain ×10, whereas the planktonic growth rate was fully restored. However, ×10 was at least as defective in biofilm formation and DNase production as P10. These data suggest that although repeated exposure to triclosan may result in an SCV-like phenotype, this is not necessarily associated with increased virulence and adapted bacteria may exhibit other functional deficiencies.

Sublethal Triclosan Exposure Decreases Susceptibility to Gentamicin and Other Aminoglycosides in Listeria monocytogenes

The human food-borne pathogen Listeria monocytogenes is capable of persisting in food processing plants despite cleaning and sanitation and is likely exposed to sublethal biocide concentrations. This could potentially affect susceptibility of the bacterium to biocides and other antimicrobial agents. The purpose of the present study was to determine if sublethal biocide concentrations affected antibiotic susceptibility in L. monocytogenes. Exposure of L. monocytogenes strains EGD and N53-1 to sublethal concentrations of Incimaxx DES (containing peroxy acids and hydrogen peroxide) and Triquart Super (containing quaternary ammonium compound) in four consecutive cultures did not alter the frequency of antibiotic-tolerant isolates, as determined by plating on 2× the MIC for a range of antibiotics. Exposure of eight strains of L. monocytogenes to 1 and 4 μg/ml triclosan did not alter triclosan sensitivity. However, all eight strains became resistant to gentamicin (up to 16-fold increase in MIC) after exposure to sublethal triclosan concentrations. Gentamicin-resistant isolates of strains N53-1 and 4446 were also resistant to other aminoglycosides, such as kanamycin, streptomycin, and tobramycin. Gentamicin resistance remained at a high level also after five subcultures without triclosan or gentamicin. Aminoglycoside resistance can be caused by mutations in the target site, the 16S rRNA gene. However, such mutations were not detected in the N53-1-resistant isolates. A combination of gentamicin and ampicillin is commonly used in listeriosis treatment. The triclosan-induced resistance is, hence, of great concern. Further investigations are needed to determine the molecular mechanisms underlying the effect of triclosan.

Evaluation of the endocrine disrupting potential of the antimicrobial triclosan on early embryonic and larval development in zebrafish.

Triclosan is a widely used chlorinated phenolic antimicrobial used in a variety of soaps, toothpastes, cosmetics, fabrics, and plastics. (Walker and Kimmel 2007). Triclosan is structurally similar to bisphenol A and dioxin, a well documented endocrine disruptor and potent carcinogen, respectively. Triclosan has recently been shown to have endocrine disrupting effects on multiple aquatic organisms and in human cells in culture, including effects on genes regulated by various estrogens, androgens, and thyroid hormones. Current concerns regarding the continued use of triclosan includes its increasing detection in urban streams and its ability to bioaccumulate in the lipid‐dense tissues of exposed organisms. Relevance to the importance of these concerns to humans includes, a report from 2008 demonstrating that 74.6% of human urine samples collected for testing contained triclosan in quantities ranging from 2.3 ug/L to 3, 790 ug/L. Triclosan, considered to be physiologically harmless, may have detrimental effects on organisms during early embryonic development. These effects may vary dramatically based on the timing of exposure. Statistically significant changes in variables such as eye diameter in zebrafish larvae can be observed when exposure times to sublethal concentrations of triclosan (200 ug/L) are prolonged through the first week of embryonic development. Preliminary results also indicate an effect on larval zebrafish heart rates. We hypothesize that this may be the result of an endocrine disrupting effect on the expression of receptors that control heart rate, such as the beta‐adrenergic and muscarinic receptors in the larval heart. Changes in several genes known to be regulated by estrogens, androgens, and thyroid hormones will be analyzed using by RT‐PCR and presented.

Triclosan Inhibits UropathogenicEscherichia coli-Stimulated Tumor Necrosis Factor-αSecretion in T24 Bladder Cellsin Vitro

Triclosan is an antimicrobial agent commonly used in consumer and medical products that inhibits bacterial fatty acid synthesis. In addition to its bactericidal effects, sublethal concentrations of triclosan reduce local inflammation, inhibit the growth of bacterial uropathogens, induce membrane stress, and inhibit P-fimbrial expression in uropathogenic Escherichia coli (UPEC). We tested whether sublethal concentrations of triclosan could reduce the adherence of UPEC to bladder and kidney cells and reduce the amount of the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) produced by these cells during bacterial challenge in vitro. Assays of bacterial growth, adhesion, and intracellularization were performed using UPEC GR12 incubated for 4 hours on monolayers of human T24 bladder cells or A498 kidney cells with various sublethal concentrations of triclosan. The expression profile of TNF-alpha from bladder cells was evaluated using ELISA. No significant decreases were observed in the adherence or invasion percentages of UPEC GR12 with either cell line when treated with sublethal amounts of triclosan. However, treatment with triclosan 0.5 microg/mL led to a significant decrease in the total number of UPEC GR12 recovered from T24 monolayers (P < 0.05). Importantly, a reduction in the expression of TNF-alpha by T24 cells was shown when UPEC GR12 was treated with triclosan (P < 0.05). Sublethal concentrations of triclosan did not inhibit the adhesion or intracellularization of UPEC into kidney or bladder cell lines but did significantly reduce the amount of TNF-alpha secreted by bladder cells. Therefore, the use of triclosan on ureteral stents may prove clinically beneficial, not only by inhibiting bacterial survival and growth within the urinary tract, but by reducing local inflammation as well.

Selection for high-level resistance by chronic triclosan exposure is not universal.

To investigate the effect of triclosan exposure on the antimicrobial susceptibilities of numerically important dental bacteria. A gradient plate technique was used to expose Fusobacterium nucleatum, Lactobacillus rhamnosus, Neisseria subflava, Porphyromonas gingivalis, Actinomyces naeslundii, Prevotella nigrescens, Streptococcus oralis, Streptococcus sanguis, Streptococcus mutans and Veillonella dispar repeatedly to escalating, sublethal concentrations of triclosan. Escherichia coli ATCC 8739 was included as an organism showing the triclosan resistance development trait. MIC values towards chlorhexidine, metronidazole and tetracycline were determined before and after biocide exposure. N. subflava, Pr. nigrescens Po. gingivalis and E. coli were highly susceptible to triclosan (MIC range 0.1-3.9 mg/L), whereas the lactobacillus and S. mutans were less susceptible (MIC range 15.6-20.8 mg/L). Triclosan exposure resulted in a highly significant ( approximately 400-fold) reduction in triclosan susceptibility (P < 0.01) for the positive control E. coli, although its MICs towards chlorhexidine, metronidazole and tetracycline were not significantly altered. Minor ( approximately two-fold) decreases in triclosan susceptibility (MIC) occurred for Pr. nigrescens and in S. sanguis and S. oralis (MBC). Mean changes in susceptibilities (MIC and MBC) of the oral species to chlorhexidine, metronidazole and tetracycline did not exceed two-fold, although chlorhexidine MBCs for S. sanguis were markedly, but transiently, increased. These data fail to demonstrate biologically significant drug resistance in triclosan-exposed bacteria and suggest that markedly decreased triclosan susceptibility, although confirmed for E. coli, is not a universal phenomenon. Other bacteria possibly possess more susceptible targets than FabI that are highly conserved, which may govern triclosan activity.