Low-Dose Agrochemicals and Lawn-Care Pesticides Induce Developmental Toxicity in Murine Preimplantation Embryos

Anne R. Greenlee; Tammy M. Ellis; Richard L. Berg


Environ Health Perspect. 2004;112(6) 

In This Article

Abstract and Introduction

Occupational exposures to pesticides may increase parental risk of infertility and adverse pregnancy outcomes such as spontaneous abortion, preterm delivery, and congenital anomalies. Less is known about residential use of pesticides and the risks they pose to reproduction and development. In the present study we evaluate environmentally relevant, low-dose exposures to agrochemicals and lawn-care pesticides for their direct effects on mouse preimplantation embryo development, a period corresponding to the first 5-7 days after human conception. Agents tested were those commonly used in the upper midwestern United States, including six herbicides [atrazine, dicamba, metolachlor, 2,4-dichlorophenoxyacetic acid (2,4-D)], pendimethalin, and mecoprop), three insecticides (chlorpyrifos, terbufos, and permethrin), two fungicides (chlorothalonil and mancozeb), a desiccant (diquat), and a fertilizer (ammonium nitrate). Groups of 20-25 embryos were incubated 96 hr in vitro with either individual chemicals or mixtures of chemicals simulating exposures encountered by handling pesticides, inhaling drift, or ingesting contaminated groundwater. Incubating embryos with individual pesticides increased the percentage of apoptosis (cell death) for 11 of 13 chemicals (p ≤ 0.05) and reduced development to blastocyst and mean cell number per embryo for 3 of 13 agents (p ≤ 0.05). Mixtures simulating preemergent herbicides, postemergent herbicides, and fungicides increased the percentage of apoptosis in exposed embryos (p ≤ 0.05). Mixtures simulating groundwater contaminants, insecticide formulation, and lawn-care herbicides reduced development to blastocyst and mean cell number per embryo (p ≤ 0.05). Our data demonstrate that pesticide-induced injury can occur very early in development, with a variety of agents, and at concentrations assumed to be without adverse health consequences for humans.

Recent epidemiologic studies suggest that parents working in areas of high pesticide application are at increased risk for adverse reproductive outcomes such as infertility (Fuortes et al. 1997; Greenlee et al. 2003; Smith et al. 1997), poor fertilization (Tielemans et al. 1999), fetal death (Arbuckle and Sever 1998; Saxena et al. 1983), and congenital anomalies (Bell et al. 2001a; Garry et al. 1996, 2002). Residential pesticide exposures and their effects on reproductive health are less well understood. A few studies suggest that maternal exposures to pesticides used around the home are associated with risk of stillbirth and fetal deaths (Bell et al. 2001b; Pastore et al. 1997; Savitz et al. 1989). Decreased birth weight and length of newborns have been associated with high levels of chlorpyrifos in plasma samples of urban minority women (Perera et al. 2003).

Timing, combinations of agrochemicals, duration of exposure, and dose may play critical roles in pregnancy outcomes. Bell et al. (2001a) reported that maternal pesticide exposures occurring during the third to eighth weeks of pregnancy have the greatest impact on fetal deaths. This temporal association strengthened when the pesticides were applied within 1 mi2 of the maternal residence. Timing of paternal pesticide exposures may also be important. Arbuckle et al. (1999a) reported that exposures to phenoxy herbicides occurring in fathers 3 months before conception doubled the risk of early spontaneous abortions in their partners.

Pesticide residues have been identified at concentrations of parts per trillion to parts per million in ovarian follicular fluid (Trapp et al. 1984), seminal plasma (Arbuckle et al. 1999b; Foster 1995), human amniotic fluid (Foster et al. 2000), fetal tissue specimens (Nishimura et al. 1977), and meconium from human neonates (Whyatt and Barr 2001). Korrick et al. (2001) and Longnecker et al. (2001) reported that the risk of preterm birth and spontaneous abortion increased with maternal serum concentrations of dichlorodiphenyldichloroethylene (DDE). Fertilization rates have been negatively correlated with levels of DDE in serum and follicular fluids of women undergoing in vitro fertilization (Younglai et al. 2002). Little is known, however, about the direct effects of pesticide contaminants on the conceptus and subsequent development near the time of implantation.

Currently, the two-generation Fertility (Reproductive) Assessment by Continuous Breeding protocol developed by the National Toxicology Program (Research Triangle Park, NC) is an accepted method for characterizing developmental and reproductive toxicants [U.S. Environmental Protection Agency (EPA) 1996]. This protocol can be used to evaluate an extensive list of abnormalities in parental and filial generations. However, it is costly and time-consuming, and it does not evaluate exposure risks encompassed by the preimplantation stage of development. The need for more rapid, comprehensive, and cost-effective tools for screening developmental toxicants has stimulated the search for in vitro methods to reduce the backlog of chemical testing critical for adequate risk assessment (National Research Council 2000).

We previously demonstrated the potential of the mouse embryo assay for identifying preimplantation toxicity induced by the estrogenic pesticide o,p´-dichlorodiphenyltrichloroethane (o,p´-DDT) (Greenlee et al. 1999). Compared with control treatment, incubation of pronuclear embryos with 0.1 µg/mL o,p´-DDT significantly reduced embryo development to blastocyst and mean cell number and increased the percentage of cells undergoing apoptotic cell death. Developmental effects were dose responsive. Furthermore, the antiestrogen ICI 182,780 abolished the developmental alterations induced by this toxicant (Greenlee et al. 2000), suggesting that the assay may be useful for characterizing injury mechanisms initiated by environmental pollutants with estrogenic activity. Implementation of the mouse preimplantation embryo assay for risk assessment purposes will require further evaluation with a variety of chemicals at ecologically relevant concentrations.

Toward this objective, we screened agricultural and lawn-care chemicals commonly used in the upper midwestern United States as single agents and as mixtures for their effects on embryo development during the preimplantation period. We hypothesized that the mouse embryo assay would prove reliable, rapid, and cost-effective for evaluating pesticide effects at low-dose concentrations and in combinations potentially encountered before a pregnancy is recognized.


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