Everyone is exposed to very low levels of pyridine in air, and food. Workers who make or use the chemical may be exposed to higher levels of it. Studies in people and animals suggest that pyridine may damage the liver. This chemical has been found in at least 11 of 1,416 national priorities list sites identified by the environmental protection agency. In vitro genotoxicity tests are employed to screen chemicals for their capability to cause various chromosomal aberrations, and the results are often used to predict their potential for carcinogenicity, however, there is controversy regarding the apparent low specificity of some in vitro genotoxicity assay, which results in positive rate. Hence, we selected both carcinogen and non – carcinogen compound. The chemical exposures for two ends – points were done simultaneously. The protocol for the two end-points was developed using the carcinogen Cyclophosphamide. The non-carcinogen chosen were pyridine. By, using human leucocytes culture both the carcinogen and non – carcinogen chemicals shows chromosomal aberration, some breaks and pulverization are obtained. The chromosome breaks and pulverization obtained both in positive and negative because both have similar toxic against the normal chromosome.
ABSTRACT
Everyone is exposed to very low levels of pyridine in air, and food. Workers who make or use the chemical may be exposed to higher levels of it. Studies in people and animals suggest that pyridine may damage the liver. This chemical has been found in at least 11 of 1,416 national priorities list sites identified by the environmental protection agency. In vitro genotoxicity tests are employed to screen chemicals for their capability to cause various chromosomal aberrations, and the results are often used to predict their potential for carcinogenicity, however, there is controversy regarding the apparent low specificity of some in vitro genotoxicity assay, which results in positive rate. Hence, we selected both carcinogen and non - carcinogen compound. The chemical exposures for two ends - points were done simultaneously. The protocol for the two end-points was developed using the carcinogen Cyclophosphamide. The non-carcinogen chosen were pyridine. By, using human leucocytes culture both the carcinogen and non - carcinogen chemicals shows chromosomal aberration, some breaks and pulverization are obtained. The chromosome breaks and pulverization obtained both in positive and negative because both have similar toxic against the normal chromosome.
KEY WORDS: Pyridine, Human leucocytes culture, Genotoxicity, Chromosome damage.
INTRODUCTION
Pyridine occurs in the environment as a by - product of coal gasification and retorting of oil shale. The compound is mobile in soil and persists in ground water near underground as a result of coal gasification, retorting of oil shale, and pesticide use. (Stuermer et. al. 1982; Sims and Sommers 1985; Sims et al. 1986; Sims and O’ Loughlin 1989). Pyridine has moderately acute toxicity and is apparently teratogenic. Because pyridine occurs in the environment and is potentially dangerous to health, and understanding of its environmental fate is important.
Pyridine is a six membered aromatic heterocycle with nitrogen as the sole heteroatom. Pyridine and related compounds are anthropogenic in nature. Once they enter the environment they may persist for long period and impart undesirable odour, taste reducing the portability of ground water. Because of its teratogenic and non - carcinogenic nature, pyridines are listed as priority pollutant by U.S EPA (Richard and Shieth.1986) and its removal from waste water prior to disposal is essential.
Human can be exposed to pyridine by inhaling it, absorbing it through the skin, ingesting it, or making skin or eye contact with it. The most important health concern for humans exposed to pyridine is damage to the liver. Other health concerns may be neurological effects, rental effects, and irritation of the skin and eyes. Pyridine can cause central nervous system depression, kidney and liver damage, and gastrointestinal upset.
MATERIALS AND METHODS Materials:
McCoy’s (Medium containing sodium bicarbonate to give a PH of 7.2 -7.4 and Streptomycin and Pencillium are an antibiotic substance) was purchased from genei Bangalore.
PREPARATION OF AB SERUM
Non citrated blood from a healthy AB donor was obtained and allowed to clot for 24 hours in the refrigerator. Clear serum was separated and inactivated at 56[0] C for 15 min. This was then filtered through Seitz filter and stored at -20 o C in small aliquots.
- Phytohemagglutinin-L (PHA-L) (a crude extract of the red kidney bean, phasealus vulgaris)
- Colchicines
Mitosis can be arrested at this stage by exposing cells to the alkaloid chemical colchicine that interferes with assembly of the spindle fibers. Such treated cells cannot proceed to metaphase until the colchicines is removed.
- Potassium Chloride And Disodium Hydrogen Phosphate
They act as the hypotonic solution which helps in the brusting of the cells.
- Giemsa Stain
The stock solution of giemsa was prepared by dissolving 1gm of giemsa powder in 54 ml of glycerol at 56 oC for 60-90 mins using a magnetic stirrer. After cooling to room temperature, 84ml of methanol was added and left on a magnetic stirrer for 1hr. It was filtered and the filterate was stored in the refrigerator.
PREPARATION OF GIEMSA STAIN (working solution)
The working solution was prepared by adding 2ml of stock solution and 2ml of 10% disodium hydrogen phosphate (Na2HPO4.2H20) to 46 ml of distilled water PH -6.8.
- Hypotonic Solution ( 0.075M)
Potassium chloride (560mgs potassium chloride in 100ml of distilled water).
- Fixative
Methanol: Glacial acetic acid (3:1 ratio).
- Cyclophosphamide(CPH) is used as positive control to check the assay system. CPH is obtained from Baxter; (German Remedies Limited).The structure of CPH is shown in fig 2.
- Pyridine
Pyridine is a colorless liquid with an unpleasant smell. It can be made from crud coal tar or from other chemicals. It is non-volatile substance. The structure is shown in fig 1
PRINCIPLE OF THE TEST METHOD
Cell cultures are exposed to the test substance both with and without metabolic activation. Predetermined intervals after exposure of cell cultures to the test substance, they are treated with a metaphase-arresting substance (eg. colcemid® or colchicines), harvested, stained and metaphase are analyzed microscopically for the presence of chromosomal aberrations.
RESULT AND DISCUSSION
In the present investigation, in pyridine was introduced in human leucocyte culture. Cyclophosphamide was taken as positive control as it is proven mutagen (schmid 1976).
STRUCTURE OF PYRIDINE
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STRUCTURE OF CYCLOPHOSPHAMIDE
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In vitro genotoxicity tests are employed to screen chemicals for their capability to cause various chromosomal aberrations, and the results are often used to predict their potential for carcinogenicity. Hence, we selected both carcinogen and non- carcinogen compound. The protocol for the two end-points was developed using the carcinogen Cyclophosphamide. The non- carcinogen chosen were pyridine (Oshiror, Piper CE, Balwierz PS, Soelter SG, 1991).
COMPARING CYCLOPHOSPHAMIDE AND PYRIDINE WITH DIFFERENT CONCENTRATION
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PYRIDINE TREATED CHROMOSOME
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- Quote paper
- Aida Emelensia (Author), P.P. Vijaya (Author), 2013, Genotoxicity of pyridine in in-vitro study by using human leucocytes culture, Munich, GRIN Verlag, https://www.hausarbeiten.de/document/213865
