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Nicotine is not clastogenic at doses of 1 or 2 mg/kg body weight given orally to male mice

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Abstract

Nicotne has been tested in the conventional mouse bone marrow assay. Single doses of 1 mg/kg bw or 2 mg/kg bw were given by oral intubations and bone marrow was sampled at 24 h (1 mg/kg) or at 6, 12 and 18 h after treatment (2 mg/kg). Nicotine treatment did not increase the micronucleus frequencies in polychromatic erythrocytes while the positive control compound mitomycin C yielded the expected result. These data contradict the only published in vivo study of nicotine in which 1.1 mg/kg bw was called positive for the induction of chromosomal aberrations in mouse bone marrow cells at all sampling intervals, even as early as 6 h after treatment. It is discussed that aberration scoring is a matter of subjectivity and depends on strict discrimination criteria between gaps and true DNA discontinuities, i.e. breaks. International collaboration has shown that micronucleus scoring is less subjective, hence more reliable. Therefore it is concluded that nocotine is not clastogenic at the doses and time intervals tested in the present experiments.

Introduction

In humans, nicotine is easily absorbed from the skin and quickly metabolised to the main metabolite cotinine, which is excreted in the urine [1], [2]. The oral LD50 for the mouse varies between 3.3 and 230 mg/kg  body weight (bw) [3]. Nicotine and its four major metabolites were not genotoxic, neither in the Ames test nor in other bacterial indicator assays [3]. In mammalian cells in vitro, positive as well as negative responses were reported for SCE and chromosome aberration induction [4], [5]. The only in vivo information on clastogenic effects of nicotine stated that nicotine-induced SCE and chromosome aberrations were reduced significantly by chlorophyllin injections in mice [6]. In this study, nicotine was given per os to male Swiss albino mice at doses of 0.77 or 1.1 mg/kg bw. A maximum of chromatid breaks (80 in 250 cells scored) was observed 6 h after treatment with the 1.1 mg/kg bw dose. Chlorophyllin was applied intraperitoneally (ip) at doses of 0.77 or 1.1 mg/kg, either 2 h before or simultaneously with the nicotine treatment, and reduced the frequencies of chromatid breaks to solvent control levels (3 in 250 cells scored). It is noteworthy that clastogenic effects observed 6 h after treatment imply that the chemical acts on cells in G2 of the cell cycle. This is a very rare event seen with so-called radiomimetic chemicals [7]. It seemed very unlikely that nicotine belongs to this class of chemicals. Therefore, we planned a micronucleus study to prove or disprove the in vivo clastogenic potential of nicotine in the low dose range tested by Sen and Sharma [6].

Section snippets

Materials and methods

The micronucleus test was performed with the method described in 1984 [8]. Each dose and control group consisted of five male (102/ElxC3H/El) F1 mice from the GSF-colony, aged 12–14 weeks, weighing between 25 and 28 g. Nicotine (CAS-No 54-11-5, Merck, Darmstadt, Germany, >99% pure) was diluted with sterile, bi-distilled water and given per os at doses of 1 or 2 mg/kg bw. Mitomycin C (MC) was injected ip at a dose of 1 mg/kg bw as positive control. The injected volumes were 0.1 ml per 10 g body weight.

Results and discussion

The results are presented in Table 1 and Fig. 1. The positive control MMC showed the expected significant increase in the frequency of micronucleated PCE (MN-PCE) [9]. Treatment with nicotine did not increase the MN-PCE frequencies in any dose or time group after oral exposure. The ratio of PCE to total erythrocytes did not indicate cytotoxic effects in any of the treated groups.

The present data are in contrast to the results published by Sen and Sharma [6] using analysis of chromosomal

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