Cytotoxicity of 8-Anilino 1-Naphthalene Sulfonic acid and its Degradation Products

 

Valli Nachiyar C*, Renugadevi K, Swetha Sunkar

Department of Biotechnology, Sathyabama University, Chennai, Tamil Nadu

 

ABSTRACT:

8-Anilino 1-Naphthalene sulfonic acid is an aromatic amine used extensively in the synthesis of azo dyes. This compound is also produced as an intermediate compound in the bacterial degradation of azo dyes during azo reduction. Moraxella osloensis, a bacterium with dye degrading ability, was checked for its ability to degrade this aromatic amine and the organism was found to be capable of degrading 64% of 20 mg l^-1 this amine. The cytotoxicity of this aromatic amine and its degradation products were checked in vitro using HEp2 (human epithelial type 2) and 3T3 (mouse embryonic fibroblast) cell lines. The results indicated the decreased toxicity of the degradation products compared to the aromatic amine. The normal cells were more sensitive to both the aromatic amine and its degradation products compared to the cancer cell lines. 

 

 

 

INTRODUCTION:

A large variety of aromatic substances participate in our life processes and their biosyyntheis and degradation form an integral part of natural carbon cycle.  Human activities are additional sources of synthetic organic chemicals in the environment. The persistence of anthropogenic aromatics in the environment has caused great concern due to their toxicity, mutagenicity and bioconcentration in higher organisms. These aromatic compounds belong to the most second abundant family of organic constituent present in the biosphere¹. Aromatic amines are widely used in the manufacture of dyes, inks, pesticides, rubber antioxidants, explosives, drugs, cosmetics, paints, varnish, lacquer, wood staining, curing agents for the synthesis of epoxy resins and polyurethanes² which makes them an indispensable group of chemicals³.

 

Peri acid and its derivatives are used extensively in dye industries for the synthesis of numerous dyes and dye intermediates. Reagent grade Phenyl peri acid is used as fluorescent probe for protein studies.

 

ANSA (N-phenyl peri acid) was reported to be one of the intermediate compounds in the degradation of Navitan fast blue S5R^4,5,6.  Many aromatic amines are recognized as known or suspect human carcinogens, and mutagenicity of aromatic amines has been demonstrated in many test systems^7,8,9. In this study an attempt has been made to find out the cytotoxicity of the ANSA before and after degradation by Moraxella osloensis, a bacterium capable degrading azo dyes isolated from textile effluent contaminate site¹⁰.

 

MATERIALS AND METHODS:

Chemicals:

All the chemicals used were of analytical grade. ANSA was purchased from Sigma. α-naphthol was purchased from SD Fine Chem. Ltd., India.

 

Microorganism and Culture Conditions:

Isolation and identification Moraxella osloensis used in this study were explained in detail elsewhere (WJMBT). For the degradation studies, Mineral salt medium having following composition was used (g/l) 1.73 K₂HPO₄, 0.68 KH₂PO₄, 0.1 MgSO₄.7H₂O, 0.1 NaCl, 0.03 FeSO₄, 1.0 NH₄NO₃, 0.02 CaCl₂.2H₂O and 5.00 Glucose. Glucose was sterilized separately and added to the medium at the time of inoculation.

 

Degradation studies:

The sterilized medium (50 ml) with the above mentioned composition containing 20 mg l^-1 of ANSA was inoculated with 2% of overnight grown bacterial culture. The flasks were incubated in an incubator shaker at 200 rev min^–1 at 30⁰C. The amount of ANSA degraded was estimated by diazotization of ANSA with a-naphthol¹¹.

 

Cytotoxicity studies:

Cell lines (Hep2 – human epithelial type 2 cells-human laryngeal carcinoma cell line and 3T3 standard fibroblast cell line) were purchased from NCCS (National Centre for Cell Sciences), Pune, India. The Cell lines were subcultured and 200 μl of cell suspension (containing 1000 cells) were transferred into 96 well plates and incubated for 24 hr to form confluency. After removing the spent media 200 μl fresh media was added. 20 μl culture supernatant containing ANSA (20 mg l^-1) as well as the degradation product of ANSA, in varying concentration were added and incubated for 4 hr. After incubation, the media was removed. 20 μl of MTT reagent (5 mg/ml) was added to each well containing media and incubated for 3.5 hr at 37°C under an atmosphere of 5% CO₂ until a purple precipitate was visible. After removing the media 150 μl DMSO (MTT solvent) was added to dissolve the purple precipitate¹². Absorbance was read at 570 nm with a reference filter of 630 nm. Percentage cytotoxicity was calculated and used for finding the IC₅₀ value of ANSA and its degradation products.

 

RESULTS AND DISCUSSION:

Moraxella osloensis is a species of Gram negative, oxidase positive, aerobic bacteria within the family Moraxellaceae in the gamma subdivision of the purple bacteria¹³. Moraxella sps. have been reported to be a potential biodegrading organism degrading variety of anthropogenic compounds¹⁴. This organism was reported to be a component of a consortium that effectively degraded Navitan Fast Blue¹⁵, and Mordant Black 14 individually¹⁰ and also as a component of Consortium¹⁶. The organism’s ability to degrade ANSA at 20 mg l^-1 was checked. After 48 h of incubation, the organism has been found to degrade 64% of ANSA.

 

The cytotoxicity studies using Hep2 and 3T3 cell lines clearly indicated that Hep2 cell line could tolerate comparatively higher concentration of ANSA and its degradation product than the 3T3 cell line. The IC₅₀ values were found to be 0.008 µg/ml and 0.022 µg/ml for ANSA and its degradation products respectively against 3T3 cell line. Hep 2 cell line required quite higher concentration of the same with the IC₅₀ values of 0.022 µg/ml and 0.74 µg/ml of ANSA and its degradation product respectively. These results clearly indicated that the toxicity of ANSA has reduced after degradation of Moraxella osloensis. These results correlate well with our previous reports on the cytotoxicity of metanilic acid against the same cell lines where the IC₅₀ values were reported to be comparatively high indicating the increased toxicity of ANSA.

 

 

Fig. 1. Cytotoxicity of ANSA and its degradation Products against 3T3

 

 

Fig.2. Cytotoxicity of ANSA and its degradation products against Hep2

 

CONCLUSION:

Moraxella osloensis, a bacterium reported for its ability to degrade Mordant Black 17, metanilic acid was also found to be capable of degrading ANSA, an aromatic amine used extensively as an intermediate in dye industries.  Media optimization has to be carried out to bring about increased degradation of ANSA which is further expected to reduce the cytotoxicity.

 

 

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14.   Anuradha, S., Karunya, A, et al “Kinetics of acid black 24 biodegradation”, J Pure Appl Microbiol, 2014, 8 (3): 2409-2415.

15.   Valli Nachiyar C, Swetha Sunkar, et al “Biodegradation of Acid Blue 113 Containing Textile Effluent by Constructed Aerobic Bacterial Consortia: Optimization and Mechanism” J Biorem Biodegrad, 2012, 3:162.

16.   Karunya A, Valli Nachiyar C, et al “Development of microbial consortium CN-1 for the degradation of Mordant Black 17” J Environ Chem Eng, 2014, 2: 832–840.

 

 

 

Accepted on 16.08.2018           © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(11): 4974-4976.