Genotoxic and cytotoxic effects of carbofuran and furadan^® on Chinese hamster ovary (CHO_K1) cells

Abstract

The in vitro geno- and cytotoxicity exerted by the N-methylcarbamate pesticide carbofuran (CF) and its commercial formulation furadan^® (F^®) were studied in Chinese hamster ovary (CHO_K1) cells by several bioassays for both genotoxicity (e.g., the sister chromatid exchange (SCE) and micronuclei (MNi) frequencies), and cytotoxicity (e.g., cell-cycle progression, mitotic index (MI), 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and neutral red (NR)). Both CF and F^® activities were tested within the range of 5–100 μg/ml. CF within a 10–100 μg/ml concentration-range induced a significant dependent increase of SCE frequency and MNi over control values. At the same concentration-range, F^® increased significantly the SCE frequencies over control values although in a non-dependent manner while only an enhanced frequency of MNi was found in those 50 μg/ml-treated cultures. No binucleated cytokinesis-block cells were found in 100 μg/ml F^®-treated cultures. The NDI index revealed a delay in the onset of cell-division with 50 and 100 μg/ml of CF and F^®, respectively. The delayed rate of nuclear division induced by 100 μg/ml of F^® was higher than that induced by an equal concentration of CF. CF and F^® induced both a significant concentration-dependent delay in cell-cycle progression and a decrease in the proliferative replication index within 5–100 μg/ml and 50–100 μg/ml concentration-range, respectively. Decreased cell viability was found in up to 26% and 47% in 100 μg/ml CF- and F^®-treated cultures, respectively. The NR and MTT assays revealed a clear cell growth inhibition when concentrations of 50 and 100 μg/ml of either CF or F^® were employed. Accordingly, the results highlight that CF by itself and F^®, even in a greater extend exerts both genotoxicity and cytotoxicity in mammalian cells in culture, at least in CHO_K1 cells.

Introduction

Carbofuran (CF) is one of the most widely granular employed N-methylcarbamate esters. CF is basically a systemic pesticide used in agriculture to control soil-dwelling and foliar-feeding insects, mites and nematodes in grain, forage, vegetable, seed and fiber crops (www.epa.gov). It is an anticholinesterase compound able to inhibit both acethylcholinesterase and butyrylcholinesterase in vivo and in vitro [1]. The main toxic effect of CF is regarded to result from carbamylation of the catalytic centre of the acethylcholinesterase in the nervous system. This results in accumulation of acetylcholine at nerve synapses and myoneural junctions leading to cholinergic signs and causing toxic effects [2]. The inhibition of the carbomoylated enzyme is labile, of short duration, and reversible compared to that induced by organophosphorus compounds, undergoing spontaneous and rapid reactivation [1], [3], [4].

In general, N-methylcarbamates have low acute toxicity to mammals, and are recognized as non-genotoxic to bacteria, yeast and fungi [5], [6], [7] as well as to mammalian cells [8], [9], [10], [11]. However, CF has been reported to be mutagenic in the Ames test after metabolic activation with S9-mix as well as in the base substitution mutation JK947 Salmonella lactam assay [12]. Furthermore, it has been claimed as a relatively weak mutagen in the Escherichia coli repair test [13]. Gentile and coworkers [6] reported the induction of unscheduled DNA synthesis in human lung fibroblasts. No single-strand breaks detected by alkaline sucrose gradient were induced in in vivo normal human skin fibroblasts [8] or mice peripheral lymphocytes [14]. However, Naravanemi and Jamil [15] reported its induction in in vitro human lymphocytes by the comet assay. The induction of cytotoxicity in human lymphocytes in vitro determined by the trypan blue dye exclusion method [15] and mitotic inhibition in mice bone marrow cells in vivo have been observed [16]. Furthermore, CF has been reported to induce sister chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells [17], and in mice [6] and rats [18] exposed in vivo, chromosomal aberrations in human lymphocytes in vitro [15] and mice in vivo [16], as well as mice sperm abnormalities in vivo [16]. Although the induction of micronuclei (MNi) has been observed for mice exposed in vivo [16], Zhou and collaborators [14] were unable to register such effect not only after CF treatment but also with carbofuranphenol, one of its metabolites. While CF induced in vitro DNA fragmentation in rat cortical neurons [19], negative results were observed in Chinese hamster lung fibroblast in vitro [20]. Furthermore, CF has been reported to be teratogenic and embryotoxic [1].

CF is recognized either as a highly toxic pesticide for human health by inhalation and ingestion, or moderately toxic by dermal absorption and when affecting nervous system functions. However, this carbamate has been classified as moderately toxic (class II) by the EPA in spite of the great variety of occupational activities that involve the use of CF around the world (www.epa.org). Due to the lack of both human cancer epidemiology and positive rodent cancer bioassays data, the IARC has not listed this N-methylcarbamate pesticide as carcinogen so far (www.iarc.fr).

Due to its widespread employment in agriculture and household, contamination of food, water and air has become serious and adverse health problem for humans, animals and wildlife. Epidemiological studies suggested that long-term exposure to CF may be associated with increased risk of gastrointestinal, neurological, cardiac dysfunction and retinal degeneration [21], [22]. Therefore, the contamination of environment with CF can easily occur, especially in those countries where it is still in use and the probability of long-term low dose exposure becomes increased. The importance of further studies on this type of pesticide to achieve a complete knowledge on its genetic toxicology seems to be, then, more than evident.

In the present study we employed the SCE, cytokinesis-block MNi frequencies, cell-cycle progression analysis, mitotic index (MI), 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and neutral red (NR) bioassays as different end-points to characterize the genotoxicity and cytotoxicity exerted on Chinese hamster ovary (CHO_K1) cells by CF and furadan^® (47% CF), one of its commercial formulation currently employed in Argentina.