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

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

Disclosures

Environ Health Perspect. 2004;112(6) 

In This Article

Discussion

Our data demonstrate that pesticide-induced injury can occur at a very early period of embryo development and at pesticide concentrations assumed to be without adverse health consequences for humans. Embryo injury was noted for single agents and for mixtures at concentrations based on 1× RfD values. The RfD is an estimate of a daily exposure to the human population assumed to be of negligible risk for deleterious effects during a lifetime. The RfD is derived by dividing the no observed adverse effect level (NOAEL) or lowest observed adverse effect level (LOAEL) dose by uncertainty factors to accommodate limitations in data, variability within humans, and differences in responses of test and target species. The use of the NOAEL/LOAEL has been criticized because of its sensitivity to sample size, high sampling variability from experiment to experiment, and the inability to use all dose-response data (Barnes et al. 1995; U.S. EPA 2000b). Future studies to evaluate risks of adverse exposures may be better served by using benchmark dose modeling because it is more inclusive of dose-response data and better reflects sample size (Castorina and Woodruff 2003).

Our findings may have implications for human reproductive health. Embryos cleaving to blastocyst yet undergoing cellular death at a higher rate could result in embryos composed of fewer cells. Unless repair mechanisms overcome cellular loss, exposures during this period could result in embryonic demise, implantation failures, or alterations in the physiologic processes underlying maternal recognition of pregnancy (Wilson 1973). Findings from animal dosing studies are consistent with these possibilities. Pregnant mice exposed to very low and low doses of an herbicide mixture (2,4-D, MCPP, and dicamba) during the period of preimplantation-organogenesis (gestation days 0-15) resulted in significant reductions in implantation sites and live births (Cavieres et al. 2002). Female mice receiving oral administration of the insecticide lindane either before or immediately after mating increased blastomere lysis and suppressed cell proliferation of two-cell embryos and morulae (Scascitelli and Pacchierotti 2003). Mice receiving subcutaneous injections of the insecticide methoxychlor on days 2-4 of pregnancy yielded embryos exhibiting suppressed blastocyst proliferation, increased percentages of nuclear fragmentation (apoptosis), and micronuclei formation (Amstislavksy et al. 2003). Embryos collected from female mice receiving a single intraperitoneal injection of the insecticide chlorpyrifos on day 0 of pregnancy showed significant increases in micronucleus formation and a dose-dependent reduction in embryo cell numbers (Tian and Yamauchi 2003). Therefore, our findings for in vitro exposed embryos closely parallel those observed for embryos collected from the reproductive tracts of mice dosed during the preimplantation period. The relevance of preimplantation embryo injury to pregnancy outcomes needs further clarification. This might be accomplished by transferring in vitro exposed embryos to foster mice and monitoring implantation rate, litter size, and pup normalcy at birth.

Agrochemicals and lawn-care pesticides were tested at concentrations ranging between parts per trillion for the insecticide terbufos to parts per billion for the herbicide metolachlor. These concentrations are environmentally relevant and physiologically achievable based on pesticide levels reported for human follicular aspirates (Baukloh et al. 1985; Jarrell et al. 1993) and for maternal and cord blood samples collected at delivery (Waliszewski et al. 2000). Comparisons between contaminant concentrations in maternal and cord plasma samples suggest a balanced state between mother and fetus with respect to circulating pesticides and metabolites (Whyatt et al. 2003). Similar correlations between maternal serum and follicular fluid contaminant levels have been reported for in vitro fertilization patients (Younglai et al. 2002). Just before ovulation, follicles become highly vascularized (Edwards et al. 1980). This increased blood flow may enhance transfer and accumulation of pollutants from serum to follicular fluids (Baukloh et al. 1985).

Adjuvants (paraffinic oils and/or surfactant mixtures) were not included in the test formulations. Adjuvants are typically combined with the active ingredients in commercial formulations to improve the characteristics of penetration, spreading, or longevity in the field (Tominack 2000). Adjuvants alone may have disruptive effects, as demonstrated by growth promotion of human tumor cell lines (Lin and Garry 2000), abnormal endocrine profiles of pesticide/adjuvant applicators (Garry et al. 1999), and cell cycle delays in embryo cleavage (Marc et al. 2002). In the latter study, pesticide toxicity was detected only in combination with a subthreshold concentration of a commercial pesticide formulation containing inert ingredients. In our study, individual agents and mixtures of agrochemicals caused measurable injury without the addition of adjuvants. It will be important to determine if embryo development is further compromised by combining pesticides with other ingredients found in commercial formulations.

Ethanol is a known teratogen. Maternal ingestion of at least 0.5 oz (14 mL) per day during pregnancy has resulted in measurable neurodevelopmental abnormalities in young children (Sood et al. 2001). This dose approximates a daily body burden of 0.01-0.03% ethanol and may vary based on the weight and genetic factors of the woman. Ethanol was used as a solvent for 11 of 13 pesticides. The 0.1% ethanol control represents the highest possible concentration of solvent. No differences in developmental parameters were measured for embryos incubated 96 hr in medium with and without ethanol (all p > 0.63). However, it is possible that ethanol supplementation at this concentration may have latent deleterious effects. Additional studies are needed to fully address this question.

Exposure to single agents and certain mixtures elevated percent cell death without affecting development to blastocyst. Other treatments stalled development to blastocyst without increasing apoptotic cellular death. For example, dicamba alone or combined with pendimethalin or 2,4-D and atrazine induced significant levels of apoptosis. However, dicamba combined with 2,4-D and MCPP significantly reduced development to blastocyst without increasing rates of cell death ( Table 2 , Table 3 , and Table 7 ). One explanation may be that early cleavage-stage embryos were not competent to initiate apoptosis. However, this is unlikely, as the requisite molecular components for the apoptotic cascade are available in the blastomeres of embryos at all stages of development (Weil et al. 1996). Another possibility to explain differing injury profiles may be the combined effects of three compounds rather than a single agent. It is believed that embryos must first differentiate into distinct embryonic regions, the ICM and the trophectoderm (TE), before apoptosis is engaged. The temporal significance of apoptosis may be to rid TE cells from the rapidly growing ICM (Pierce et al. 1989). In support of this possibility, Hardy (1999) and Brison and Schultz (1997) noted that most apoptotic activity was confined to the ICM region of blastocyst embryos. We also localized apoptosis primarily in the region of the blastocyst ICM (Figure 2). Therefore, embryos may need to cleave normally to blastocyst to demonstrate an increased vulnerability to low-dose contaminants. More substantial injuries, causing embryos to stall differentiation to ICM and TE, would result in rates of apoptosis similar to the negative control treatment.

Agrochemicals and lawn-care pesticides chosen for testing are those still commonly used in the upper midwestern United States. Mixture formulations were based on possible exposures routes (e.g., ingesting contaminated groundwater; mixing and handling pesticides; inhaling pesticide drift). Compounds could also be screened based on common mechanisms of pesticide action. The embryo model is well suited for accommodating both approaches.

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