Triclosan Effects on Lungs

Introduction 

Triclosan (TCS) as the biocide is commonly used in household products and various oral hygiene brands including detergents, freshener in addition to antibacterial soaps. Therefore, it is clear that most people are exposed to triclosan, and it is commonly found in urine and breast milk causing environmental hazard. According to Adolfsson-Erici et al, (2002), the triclosan’s antibiotic mechanism is commonly the inhibition of enoyl-acyl carrier protein reductase which is normally related to bacterial lipid biosynthesis. Most of the previous results indicated that the inhaled triclosan-induced pulmonary toxicity, especially in rats. In the recent times, most of the products including deodorant and air freshener are commonly used in most homes to remove odor (Jones, et al, 2000) . However, these products have been shown to have a high level of spread of inhalable particles such as nano-size and the inhalation exposure especially in most indoor environments hence affecting lungs (Kepner, 2004) . Research has pointed out that people are highly exposed by inhalation to triclosan often by the use of deodorant and air freshener containing triclosan. Triclosan, therefore, can cause pulmonary toxicity. The primary purpose of the research was to evaluate triclosan effects on lungs. 

Methods 

The following materials were used in the experiment: triclosan and reagents, triclosan stock solution prepared in dimethylsulfoxide in addition to male sprague-dawley transgenic mice that express human’s mutant form. 

Intratracheal instillation 

20 transgenic mice of about five weeks old were grouped into three major controlled groups. Each of the rats was given triclosan intratracheal instillation where the lowest group was given about ten μg/B.W. kg, and the highest group was given about 1,000 μg/B.W. kg. Further, the exposure doses used in the experiment took into consideration acute, low dose, and high dose exposure. On the other hand, the controls in the experiment were also exposed to approximately 0.5% DMSO’s saline as a vehicle. In addition to this, about half of all the rats in the three groups were critically examined in order to verify the bronchoalveolar lavage fluid. The rest of the transgenic mice used in the experiment were also inspected histopathologically, and this used the routine methods. After intratracheal instillation, these rats were then sacrificed after two weeks. 

BALF analysis 

The transgenic mice were anesthetized using an overdose of isoflurane. Further, using repeated whole-lung lavage, BALF was acquired. The rat’s left lung was further clamped off while the right lung-lavaged thrice using calcium of about 3ml and solution of magnesium free phosphate buffer with about pH 7.4. Further, the acquired samples were then centrifuged for about ten minutes at approximately 1,500 rpm. The first lavage’s cell-free supernatant was set aside from the rest of the samples used in the experiment specifically for biochemical analyses. On the nother hand, the recovered cells from the all of the lavages were then re-suspended in the PBS. After that, there was the counting of the total cells in BALF by the use of Vi-Cell ® XR analyzer. Additionally, there was the performance of cell differential on cytospin preparations which were stained using Diff-Quick staining. By the use of light microscopy, the BALF cells were counted as Polymorphonuclear leukocytes. BALF supernatant’s total protein was later quantified by the use of BCA protein assay kit and further, BALF lactate acid dehydrogenase activities were measured by the use of QuantiChrom Lactate Dehydrogenase Kit. Further, BALF’s tumor necrosis factor alpha and interleukin-6 were approximated by the use of enzyme-linked immunosorbent assay kits. 

Cytotoxicity 

The triclosan’s cytotoxicity was later on measured by the use of 3-(4, 5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay. The L2 cells which comprised of the Korea cell bank, rat lung epithelial cells in addition to Seoul and Korea were then seeded in approximately 96-well plates at an about 1.0 × 10 cells density per well later incubated overnight. The incubation of the cells was for about 4 hours and 24 hours but with varying of triclosan. After incubation, 5 mg/ml in PBS solution of approximately ten μl of MTT was to the entire well then supertant was removed. Additionally, dimethyl sulfoxide of about 100 μl was added. Further, the absorbance rate was later on measured by the use of the micro-plate reader and this done at a wavelength of approximately 540 nm. 

Statistical analysis 

Diverse Student’s t-test and the variance of analysis were applied in this experiment to compare the exposure groups with control groups specifically. Further, the significant level was P< 0.05 and P< 0.01. 

Results 

Based on the experiment, the triclosan’s that intratracheal instillation induced resulted in an increased BALF’s pulmonary toxicity marker, and this was after a day of exposure. Further, the index levels were however recovered throughout the convalescence period of about 14 days. Triclosan’s treatment was further reduced cell viability with the morphological change in the L2 cells. It was further established that pulmonary inflammation often comes with an early immune response, and this is commonly informed of activation of the alveolar macrophages. Additionally, pulmonary inflammation led to an increase in the PMN percent within the BALF cells. BALF cytology analysis in this experiment established that the administration of triclosan’s intratracheal instillation led to an increased count of the total cells and the PMNs’ percentage in the BALF cells, and this was after a day of post-exposure. 

There was a significant increase in the TP’s concentration within the BALF exposed to the triclosan than those from the control groups. The analysis of LDH established no significant change when comparing to the control group. The research of the experiment further showed a temporal increase in the secretions of the TNF-α and IL-6 specifically at the exposure group that was recovered immediately the elapse of two weeks. The lung epithelial cells that had been exposed to the triclosan led to a significant reduction in the cell viability. The IC values were approximated to be between 188.58 μM and 118.86 μM, and this was after the 4 and 24 hours treatment respectively. 

Pulmonary toxicity of triclosan 

The figures A and B below show BALF’s cytological characterization immediately after the triclosan’s intratracheal instillation. 

Figure A 

TC 

In 

BALF 

(% of control) 

Figure A shows the total cells count in BALF 

F igure B 

Percent 

Of 

PMNS 

(% of control) 

Figure B shows percentage of the BALF cells’ polymorphonuclear neutrophils. 

The Figures C and D shows the biochemical markers’ analysis in the bronchoalveolar lavage fluid. 

Figure C 

TP 

in 

BALF 

(% of control) 

Figure C shows total protein concentration change in the BALF. 

F igure D 

LDH 

in 

BALF 

(% of control) 

Figure D shows BALF’s lactate dehydrogenase level. 

The two figures E and F show the concentration of cytokine’s secretion in the BALF immediately after the exposure of the triclosan. 

Figure E 

TNF-α 

in 

BALF 

(% of concentration) 

Figure E shows tumor necrosis factor alpha (TNF-α) concentration in the BALF. 

Figure F 

IL-6 

in 

BALF 

(% of control) 

Figure above shows Level of interleukin-6 level (IL-6) in the BALF. 

Discussion 

The result of the experimenter, therefore, pointed out those macrophages and the PMNs are vital since they play a significant role regarding protecting the lungs especially during biocide’s pulmonary early exposure. TP level within the BALF showed a significant alveolar or capillary membrane barrier in the lungs while the LDH level showed cytotoxicity marker barrier in the lungs (Kepner, 2004) . Focusing on the result of the experiment, it can be argued that triclosan has a significant effect on lungs, and this is where it induces an early inflammatory response which then results in an acute lung injury (Jones, et al, 2000) . To establish the triclosan’s pulmonary toxicity after instillation, I analyzed triclosan’s inflammation response in the lungs by the use of BALF analysis. 

Further, based on these outcomes, the triclosan’s intratracheal instillation have a significant effect on the PMNs recruitment as an acute lung injury (Abraham, 2003) . The outcome of the experiment was obtained after a period of two weeks after the instillation; however, the TP level of concentration increased significantly, and this pointed towards the direction that triclosan has a significant effect on lung function. Most of the previous results had indicated that a continuous inhalation of the triclosan increased respiratory system’s focal ulcerations and acute inflammation morality level (Calafat et al, 2008) . The results of the experiments can be said to be closely corresponding with most of the previous research carried out using DDAC in mouse lung fibroblasts. 

Conclusion 

The research focused on the triclosan effects on lungs. The results established that triclosan exposure by the intratracheal instillation had a significant effect on lungs by causing an induced acute inflammation which affected the ability of lung functionality. There is the need for repeated investigation of triclosan effect on lungs to confirm these results, and this should focus on the analysis of molecular level for the pulmonary toxicity’s mechanism. 

References 

Abraham, E. (2003). Neutrophils and acute lung injury. Critical care medicine , 31 (4), S195. 

Adolfsson-Erici, M., Pettersson, M., Parkkonen, J., & Sturve, J. (2002). Triclosan, a commonly used bactericide found in human milk and in the aquatic environment in Sweden. Chemosphere , 46 (9), 1485-1489. 

Calafat, A. M., Ye, X., Wong, L. Y., Reidy, J. A., & Needham, L. L. (2008). Urinary concentrations of triclosan in the US population: 2003-2004. Environmental health perspectives , 116 (3), 303. 

Jones, R. D., Jampani, H. B., Newman, J. L., & Lee, A. S. (2000). Triclosan: a review of effectiveness and safety in health care settings. American journal of infection control , 28 (2), 184-196. 

Kepner, J. (2004). Triclosan hazards continued. Pesticides and You. Winter , 2005 (24), 4.