DNA Toxicity of Cocaine Hydrochloride and Cocaine Freebase by Means of DNA Image Analysis on Tetrahymena pyriformis.

Abstract

The popularity of cocaine boomed in the 1980s when freebase cocaine (crack) became widely known. The drug can be consumed as both salt and freebase either by sniffing or smoking.1) The ability to achieve rapid, high brain levels of cocaine by smoking crack has greatly increased its abuse potential and also its toxicity.2) Cocaine in the form of freebase is preferred by drug misusers because it is much more socially acceptable, particularly among habitual smokers of marihuana.3) At the same time, freebase appears to be much cheaper than cocaine hydrochloride. 4,5) The toxic effects of cocaine in humans are similar to those of crack, 6) although the signs and symptoms caused by crack are more intense and short-lasting. 7,8) The protozoan Tetrahymena pyriformis, a monocellular organism, is a suitable experimental model for functional, pharmacological, toxicological or immunotoxic studies. [9][10][11][12] Our experience with this protozoan has led us to consider its DNA content as a parameter that we could examine for the detection of toxic responses to cocaine. This protozoan contains a macronucleus which participates in the conjugation and division of the cell and contains deoxyribonucleic acid. In most strains of Tetrahymena, a micronucleus is also present.13) Macronucleus of the protozoan Tetrahymena pyriformis contains 12-24 times as much DNA as does micronucleus and practically represents the total DNA content of the cell. Macro-and micronuclei both give strongly positive cytochemical reactions for DNA (Feulgen). 14,15) The aim of this study was to investigate the possible toxic effects of cocaine salt and cocaine freebase (crack) on the DNA content of the macronuclei of the protozoan Tetrahymena pyriformis, in an effort to explain the underlying mechanisms for the immunodeficiency which cocaine addicts develop, since our daily practice concerns drug addicts. MATERIALS AND METHODSA computerized image analysis system (CIAS), based on high-resolution scanning, has proved to be an effective approach to examine and classify stained cells spread on a glass slide in the form of smears. 16,17) This technique is based on a conversion of optical images of cells into arbitrary numerical values (digitization) based on light absorbance or transmission.The CIAS was used to study the macronuclei of the cell. Relevant procedures with the protozoan Tetrahymena pyriformis have been established for years. 18,19) This protozoan was grown axenically in a proteose-peptone broth at 25°C, without stirring the cultures, until reaching late log phase, generally 4-5 d after inoculation. 12)Cocaine hydrochloride or crack was added to the 100-ml cultures as aqueous solution in doses 1 and 2 mg of substance per 100 ml of culture respectively. These doses were selected based on previous studies carried out in our laboratory with doses ranging from 0.15 to 0.60 mg of both cocaine and crack per ml of protozoan culture, which did not produce any effects.12) The toxic effects of cocaine in humans have been noted with blood concentrations rangi...

Effect of cocaine and crack on the ploidy status of Tetrahymena pyriformis: a study using DNA image analysis

“…The quantification of nuclear DNA is increasingly coming into practice in both research and clinical application (Cohen 1996;Roels et al 1996;Stefanidou et al 2002). Among the various stains and chromogenic reagents for quantitative staining of DNA have been recommended in literature, Feulgen reaction is one of the most widely accepted staining method for DNA cytometry (Cohen 1996;Roels et al 2000).…”

“…Cocaine hydrochloride or crack was added each separately to a 100-mL culture, as aqueous solutions, at doses of 0.5, 1 and 2 mg (5, 10 and 20 lg per mL culture). These doses were selected based on previous studies carried out in our laboratory and are considered as acutely non-toxic (Stefanidou et al 1999(Stefanidou et al , 2002. For each drug and dose selected one protozoa culture was tested and another culture served as control.…”

“…A computerized image analysis system (CIAS), based on high resolution scanning, has proven to be an effective approach to examine and classify stained cells spread on a glass slide in the form of smears (Stefanidou et al 2002(Stefanidou et al , 2008. This technique is based on a conversion of optical images of cells into arbitrary numerical values (digitization) based on light absorbance or transmission.…”

“…Previously, the DNA content of T. pyriformis macronuclei after exposure to cocaine and crack has been measured using DNA image analysis system where it was shown that both drugs affect the DNA content of the protozoa macronuclei (Stefanidou et al 2002). The fluctuations in the DNA content of T. pyriformis macronuclei after exposure to both drugs observed previously may result from a possible effect of the drugs on chromosome segregation during cellular division.…”

“…The protozoon Tetrahymena pyriformis is an established experimental model in biological studies (Sauvant et al 1999) and has been extensively used as a model organism to test the toxicity of different substances using several endpoints, such as phagocytic activity and DNA content (Stefanidou et al 2002(Stefanidou et al , 2008. Previously, the DNA content of T. pyriformis macronuclei after exposure to cocaine and crack has been measured using DNA image analysis system where it was shown that both drugs affect the DNA content of the protozoa macronuclei (Stefanidou et al 2002).…”