Mutation Research 521 (2002) 165–178 Micronucleus frequency and proliferation in human lymphocytes after exposure to herbicide 2,4-dichlorophenoxyacetic acid in vitro and in vivo Nina T. Holland a,∗ , Paurene Duramad a , Nathaniel Rothman b , Larry W. Figgs c , Aaron Blair b , Alan Hubbard d , Martyn T. Smith a a Division of Environmental Health Sciences, School of Public Health, University of California, Berkeley, CA 94720-7360, USA b National Cancer Institute, Bethesda, MD 20892, USA c St. Louis University, St. Louis, MO 63108-3342, USA d Division of Biostatistics, School of Public Health, University of California, Berkeley, CA 94720-7360, USA Received 2 January 2002; received in revised form 15 August 2002; accepted 21 August 2002 Abstract Widespread use of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) and its association with non-Hodgkin’s lymphoma (NHL) and other cancers has raised public concern. Here, micronucleus (MN) formation has been used as a biomarker of genotoxicity, and replicative and mitotic indices (MIs) as biomarkers of cell cycle kinetics in human lymphocytes. Cells were cultured either as whole blood or isolated lymphocytes and treated with pure or commercial forms of 2,4-D at doses between 0.001 and 1 mM for 48 h. Exposure to 2,4-D produced a minimal increase in MN in whole blood and even smaller one in isolated lymphocyte cultures. This induction took place only at levels approaching cytotoxicity and was accompanied by a significant inhibition of replicative index (RI). At a low (0.005 mM) dose of commercial 2,4-D, a small, marginally significant increase in RI (12–15%) was found in two independent sets of experiments (P = 0.052). Additionally, we found that lymphocyte RI was more affected by commercial 2,4-D containing 9.4% of the chemically pure 2,4-D, than with an equal concentration of the latter suggesting that other ingredients present in the commercial pesticide may be responsible or may enhance the effect of 2,4-D. Mitotic index, however, did not show any significant change with either commercial or pure 2,4-D. The lymphocytes of 12 male applicators exposed solely to 2,4-D during a 3-month period had a significantly higher RI than the same group prior to exposure and than a control group (P< 0.01), in accordance with the in vitro finding of increased RI at low doses. © 2002 Elsevier Science B.V. All rights reserved. Keywords: 2,4-D; Human lymphocytes; Applicators; Replicative index; Micronucleus frequency 1. Introduction 2, 4-Dichlorophenoxyacetic acid (2,4-D) is a phe- noxy herbicide that functions by maintaining high ∗ Corresponding author. Tel.: +1-510-642-8781; fax: +1-510-642-0427. E-mail address: ninah@uclink4.berkeley.edu (N.T. Holland). levels of the plant hormone auxin, resulting in over- stimulation of plant growth and ultimately death. 2,4-D is currently the most widely-used herbicide in the world, with almost 60 million pounds being used annually in the US. [1]. A two- to eight-fold increase in the incidence of non-Hodgkin’s lymphoma (NHL) among farmers who frequently used phenoxy herbi- cides such as 2,4-D has been reported among farmers 1383-5718/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII:S1383-5718(02)00237-1