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

Materials and Methods

All experiments were reviewed and approved by the Marshfield Clinic Institutional Animal Care and Use Committee. Experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council 1996).

CD-1 female mice 21-26 days of age (Charles River Laboratories, Portage, MI) were superovulated with intraperitoneal injections of 5 IU follicle-stimulating hormone (Gestyl; Professional Compounding Center of America, Inc., Houston, TX) followed by 10 IU human chorionic gonadotropin (hCG; Schein Pharmaceutical, Inc., Florham Park, NJ) 47 hr apart. Females were housed with proven CD-1 male mice.

Embryos were collected from the oviducts of female mice with vaginal plugs 18 hr after hCG injection. Reproductive tracts were placed in 37°C modified Earle's balanced salt solution (EMG) (Scott and Wittingham 1996) containing 0.3% bovine serum albumin (BSA; A3311), 0.5 mM glucose (G6152), 1.0 mM glutamine (G1146), 0.05 mM EDTA (E4884), 21.4 mM lactate (L7900), and 0.33 mM pyruvate (P4562) (all from Sigma Chemical Co., St. Louis, MO) and transported to the laboratory in a portable CO2 incubator (K Systems, Birkerad, Demark). Pronuclear (one-cell zygote) embryos were teased out of the ampullae, and cumulus masses were removed by a 3- to 5-min incubation in 0.2 mg/mL hyaluronidase (H3506) in EMG plus BSA. Embryos were washed through three 3-mL rinses of EMG plus BSA and two 25-µL rinses of EMG without BSA before transferring 20-25 embryos to 25-µL drops of EMG without BSA containing 0.1% vol/vol ethanol (negative injury control), EMG without BSA with no ethanol (solvent control), 0.1 µg/mL o,p´-DDT (positive injury control), individual pesticides, or pesticide mixtures. Embryos were handled in low light using conditions that minimized pH, osmotic, and temperature fluctuations. Microscope stages were heated.

One-cell, pronuclear embryos were incubated with agricultural and lawn-care chemicals at very low-dose concentrations based on the 1× reference dose (RfD) value for each chemical as reported by Kamrin (1997) and the U.S. EPA (2000a). The RfD is an estimate of a daily oral exposure to the human population (including sensitive subgroups) that is likely to be without an appreciable risk of deleterious effects during a lifetime (U.S. EPA 2000a). It is derived from the highest dose level of a chemical that has no adverse effect in LD50 animal studies (based on the dose that is lethal to 50% of study animals) divided by a safety factor, typically 100 (Kamrin 1997). RfD units are expressed as milligrams per kilogram of body weight per day. For our experiments, RfD units were converted to milligrams per milliliter, micrograms per milliliter, or nanograms per milliliter because embryos were incubated 4 days in vitro with pesticides and fertilizer diluted in culture medium. Cultures were not replenished with pesticides during the incubation period because paracrine factors synthesized by neighboring embryos are essential for optimal embryo development in vitro (Brison and Schultz 1997).

The working concentrations of the controls and agrochemicals and the percent purity of the agents are shown in Table 1 . Chemical purity was determined by the manufacturers using gas chromatography, mass spectroscopy, flame ionization, or titration. The agrochemicals and lawn-care pesticides tested were those most commonly used in the upper midwestern United States, including six herbicides [atrazine, dicamba, metolachlor, 2,4-dichlorophenoxyacetic acid (2,4-D), pendimethalin, and mecoprop (MCPP)], three insecticides (chlorpyrifos, terbufos, and permethrin), two fungicides (chlorothalonil and mancozeb), one drying agent (diquat), and one fertilizer (ammonium nitrate). Pesticides and desiccant were purchased from AccuStandard, Inc. (New Haven, CT), and ammonium nitrate was purchased from Sigma Chemical Co. Mixtures were prepared using combinations of agrochemicals to simulate preemergence and postemergence (before and after plants break the surface of the ground) herbicide formulations, a fungicide-desiccant combination, groundwater contaminants, insecticide combination, and lawn-care herbicides.

Stock concentrations of agrochemicals were prepared in 100% ethanol (AAPER Alcohol and Chemical Company, Shelbyville, KY). Working concentrations were prepared by serially diluting 10,000× stock solutions to 1× working solutions in EMG without BSA. Stock and working dilutions of mancozeb and diquat were prepared in tissue culture medium because they were more soluble in water than in organic solvents.

Two negative control treatments were prepared by supplementing EMG without BSA culture medium with or without 0.1% vol/vol ethanol. Results of the negative injury controls were compared to determine possible developmental effects of the solvent (ethanol) and to identify pesticide treatment effects. The positive injury control treatment was prepared by supplementing EMG without BSA with 0.1 µg/mL o,p´-DDT (AccuStandard, Inc.). We selected this pesticide and dose because the treatment reliably induces developmental injury in preimplantation embryos (Greenlee et al. 1999). Results from the negative and positive injury controls provided measures of intra- and interassay variation.

At the end of the 96-hr culture period, embryos incubated in test and control treatments were scored for development to blastocyst (Gerrity 1988), percentage of apoptosis, and mean cell number per embryo (Brison and Schultz 1997; Greenlee et al. 1999). Development to blastocyst was determined by identifying the percentage of embryos at the one- to eight-cell, morula, blastocyst, expanded, and hatched blastocyst stages using a Nikon Diaphot inverted microscope fitted with Hoffman differential contrast optics (Modulation Optics Inc., Greenvale, NY) and magnification of 100×. We used the following formula to calculate the percentage of embryos developing to the blastocyst stage:

Percentage of embryos ≥ Blastocyst
= (No. of embryos ≥ Blastocyst × 100)
÷ Total no. of embryos in culture drop.

Photomicrographs were taken at a magnification of 200× with a Nikon N2000 camera (Nikon) and Kodak Ektachrome ASA 400 film (Eastman Kodak, Rochester, NY). Digital images presented in Figures 1 and 2 were prepared from scanned photographs.

Photomicrograph showing development of embryos cultured 96 hr with (A) negative injury control 0.1% (vol/vol) ethanol, (B) positive injury control 0.1 µg/mL o,p´-DDT, (C) 1× RfD dicamba, (D) 2,4-D, (E) MCPP, or (F) a mixture of dicamba, 2,4-D, and MCPP. Magnification, 200×. Red arrows in (A) point to embryos at the blastocyst, expanded blastocyst, and hatching blastocyst stages of development. Lavender arrows in (B) and (F) point to embryos stalled at earlier cleavage stages (two-cell, four- to eight-cell, and morula).

Single embryos from each of the treated groups stained for apoptosis after embryos were cultured 96 hr with (A) negative injury control 0.1% (vol/vol) ethanol, (B) positive injury control 0.1 µg/mL o,p´-DDT, (C) 1× RfD dicamba, (D) 2,4-D, (E) MCPP, or (F) a mixture of dicamba, 2,4-D, and MCPP at 1× RfD concentrations. Magnification, 400×. Apoptotic nuclei stain yellow-green and viable cell nuclei stain orange-red. White arrows point to the ICM region of the embryo containing the area of highest apoptotic activity.

We determined the percentage of embryo blastomeres undergoing cell death by apoptosis using the terminal deoxynucleotidyl transferase-mediated 2´-deoxyuridine 5´-triphosphate (dUTP)-biotin nick end-labeling (TUNEL) assay. Blastocysts were fixed overnight in 25-µL droplets of 3.7% paraformaldehyde in phosphate-buffered saline (pH 7.3) (Sigma Chemical Co.) covered with mineral oil (M8410; Sigma Chemical Co.) at 4°C. Fragmented DNA was quantified by labeling the 3´-OH ends of DNA with fluorescein-conjugated dUTP (Apoptosis Detection System; Promega, Inc., Madison, WI). Cell nuclei were counterstained by incubating embryos 20 min in 0.1 mg/mL propidium iodide. Stained embryos were placed in 3-µL drops of Vectashield fluorescence mounting medium (Vector Laboratories, Inc., Burlingame, CA) on numbered glass slides. The test treatments were keyed and examined by a single technician. Cell nuclei were counted at 400× using a Nikon Optiphot-2 microscope fitted with a DAPI/FITC/Rhodamine triple-band pass filter (Nikon). The nuclei of apoptotic cells stained yellow-green, whereas propidium-iodide-stained nuclei of viable cells appeared orange-red. The cell number per blastocyst was determined by combining the counts of orange and green nuclei per embryo. The total number of stained nuclei per embryo was initially counted twice. A third determination was performed if the previous two counts differed by > 5%. The median of the resulting two or three counts was automatically calculated by the database used in the analysis. The percentage of apoptosis was calculated by dividing the number of green nuclei by the total number of nuclei per embryo and multiplying by 100.

Analyses of the primary outcome measures (percentage of embryos developing to blastocyst, the percentage of cells per embryo undergoing apoptosis, and the mean cell number per embryo) were based on analysis of variance (ANOVA) for mixed linear models (SAS Institute Inc. 1997). Experimental replicates, which included negative injury or solvent control, o,p´-DDT, and a subset of pesticide and dose combinations (because of limitations on the number of embryos available at a given time) were modeled as a random effect. Batches of embryos served as the unit of analysis, with a mean of 22 embryos per treatment (including control) for the percentage developing to blastocysts and a mean of 13.5 embryos for the percentage of apoptosis and the cell number. Analyses were weighted in proportion to the number of embryos used for a given treatment. This weighted-least-squares approach assumes that observations based on more embryos have lower variability and weights them optimally in the analysis. Each treatment appeared in at least four experiments, all of which included negative and positive controls for reference. Treatment means were computed from the statistical model to incorporate this weighting and to adjust for differences in experimental replicates. As planned by design, each treatment was compared with the control, and the results in this report are deemed statistically significant at the 5% level (p < 0.05) without adjustment for multiple comparisons.


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