Micronucleus frequency in human lymphocytes after exposure to diphenylamine in vitro

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Abstract

Diphenylamine (DPA) is an antioxidant compound that occurs naturally in several vegetables. It is widely applied in agriculture for preservation of the quality of apples and pears, and used for controlling superficial scald, a disorder that renders fruits of a number of apple cultivars unfit for the market. Because of its anti-oxidative properties, DPA also has several industrial applications. The potential genotoxic effect of DPA on human lymphocytes has previously been investigated in only two studies, which focused on detection of chromosome aberrations and sister chromatid exchange, respectively. In the present analysis, we evaluated micronucleus (MN) formation in freshly isolated human peripheral lymphocytes exposed to different concentrations (0.625, 1.25, 2.50, 5.0 and 10.0 μg/ml) of DPA. Peripheral venous blood was collected from ten healthy subjects, and a total of 10,000 bi-nucleated cells were analyzed. Results indicated that DPA significantly increased the micronucleus frequency at concentrations of 1.25 μg/ml and higher. Significant differences in the MN frequency were also found between the lower dose (0.625 μg/ml) and all other doses tested, with the exception of 1.25 μg/ml. Our results indicate a potential cytogenetic effect of DPA on human cells in vitro and require further in vivo studies to clarify the actual genotoxicity of this compound and the consequent risks for human health.

Introduction

Diphenylamine (DPA) is an antioxidant widely used for preservation of the quality and prevention of post-harvest deterioration of apples and pears, in particular for controlling superficial scald, a disorder that renders fruits of a number of apple cultivars unfit for the market. Because of its anti-oxidative properties, DPA also has several industrial applications, for example as a precursor of dyes, pharmaceuticals, photographic chemicals, and as a stabilizer in nitrocellulose-containing explosives and propellants (reviewed in [1]). Moreover, DPA is a natural compound found in some vegetables, such as onions [2], baked potatoes [3], buckwheat flour [4], black and green tea [5], and dried plums [6].

Eco-toxicological studies indicate that DPA and its derivatives are contaminants in soil and water, and potentially hazardous to aquatic organisms [1]. DPA treatment in short-time studies and during long-term expositions in animal experiments showed an increase of liver- and kidney-damaging effects [7], [8], [9]. Lorenzin [10] assessed the amount of pesticide residues in Italian pre-packed meals: DPA was shown to be one of the pesticides most frequently found in fruit and side dishes. Cytogenetic monitoring of human populations exposed to pesticides is generally based on the analysis of the genotoxic effects induced by chronic low doses of complex chemical mixtures. As a consequence, it is often very difficult to associate the observed genotoxic damage to specific chemical classes or compounds. On the other hand, in vitro and in vivo studies focusing on single substances provide more informative evidence about the genotoxic effects of specific pesticides.

Unfortunately, in vitro experiments with DPA are scanty. Dolara and co-workers [11], [12] tested the effects of a mixture of 15 pesticides commonly found in Italian foods (including DPA at 14.4%) on human lymphocytes. Results indicated that the pesticide mixture did not induce significant variations in chromosome number or the frequency of chromatid aberrations. Conversely, a moderate but statistically significant increase of sister-chromatid exchange (SCE) at low concentrations of the pesticide mixture was observed. Subsequently, Ardito et al. [13], analyzing the effects of DPA on human lymphocytes, observed a significant increase of SCE frequency only at high concentrations.

In a FAO-WHO joint meeting [14], on the basis of negative results from studies conducted in vivo on different animals, it was concluded that although DPA may have genotoxic potential, it is unlikely to be a human genotoxic hazard. In the same meeting, the recommended maximum residue limit (MRL) on apples was 5 mg/kg, while the acceptable daily intake (ADI) for humans for this compound was set at 0–0.08 mg/kg bw [14], [21].

In this study we analyzed the effects of different DPA concentrations on human lymphocytes, by use of the micronucleus (MN) assay. Micronuclei are either a-centric chromosome fragments or whole chromosomes left behind during mitotic cell division, and appear as small additional nuclei in the cytoplasm of interphase cells. In contrast to analyses of CA and SCE, which mainly reveal alterations in chromosome structure, the MN assay – in combination with centromere analysis – may allow detection of both clastogenicity (chromosome breakage) and aneugenicity (chromosome lagging due to dysfunction of the mitotic apparatus) [15], [16], [17], [18].

The aim of the study was to determine whether DPA, at different concentrations, could induce genotoxic damage in cultured human lymphocytes. We tested 0.625, 1.25, 2.5, 5 and 10 μg/ml of DPA, where 0.625, 1.25, and 2.5 μg/ml represent the sub-multiple and 10 μg/ml the multiple of the MRL, which is the value accepted for apples (5 μg/ml).

Section snippets

Chemicals, media, and enzymes

Diphenylamine (DPA) (IUPAC name, N-phenylbenzenamine; CAS nr 122-39-4) was obtained from Labservices, Bologna, Italy and dissolved in DMSO (CAS no. 67-68-5). Gibco RPMI 1640 cell-culture medium supplemented with l-glutamine, foetal calf serum, phytohaemagglutinin (PHA), and antibiotics were purchased from Invitrogen-Life Technologies, Milan, Italy. Cytochalasin-B and mitomycin-C (MMC) were obtained from Sigma–Aldrich, Milan, Italy. Methanol, acetic acid, Giemsa staining solution, and

Results

To assess the effects of DPA on human lymphocytes, the MN frequency was investigated at different concentrations of the pesticide (Table 1). The results indicate that DPA significantly (P < 0.05) increased the MN formation compared with the negative control, at all concentrations (Table 1). When compared with the solvent control (1% DMSO), DPA significantly increased MN frequency at all concentrations with the exception of 0.625 μg/ml. Moreover, highly significant (P < 0.001) differences in MN

Discussion

A large body of literature has been devoted to the analysis of possible toxic effects of pesticides with different animal models and cellular systems. Although various experimental data have provided convincing evidence that pesticides can exert mutagenic/genotoxic effects in vivo and in vitro, detailed information about aneugenic and clastogenic effects of exposure to specific pesticides is limited and sometimes inconsistent. Among the cytogenetic end-points (CA, SCE, MN) commonly used to

Conflict of interests

The authors declare that they have no conflict of interests.

Acknowledgements

We are grateful to Natascha Rogge for her valuable help and criticism and to all volunteers who participated in this study. The authors are also thankful to the anonymous referees for their helpful comments and constructive suggestions. This work was supported by grants from the Italian Ministry of University and Scientific Research.

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