Research report
Chlorpyrifos exposure during a critical neonatal period elicits gender-selective deficits in the development of coordination skills and locomotor activity

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Abstract

The widespread use of chlorpyrifos has raised concern about the potential consequences of fetal and childhood exposure. Previous studies have shown that apparently subtoxic doses of chlorpyrifos are nevertheless capable of affecting brain development by inhibiting mitosis, eliciting apoptosis, and altering neuronal activity and reactivity. To determine whether these biochemical changes elicit behavioral abnormalities, we evaluated coordination skills and open field behaviors in developing rats. Administration of 1 mg/kg s.c. of chlorpyrifos on postnatal (PN) days 1–4 elicited deficits in reflex righting on PN3–4 and in geotaxic responses on PN5–8, an effect that was specific to females. However, the ontogeny of more complex behaviors indicated a subsequent selectivity toward males. In the periweaning period, open-field locomotor activity and rearing were markedly reduced in male rats that had been exposed to chlorpyrifos on PN1–4, whereas no effect was detected in females. The gender-selective behavioral effects were associated with greater sensitivity of males to inhibition of cholinesterase in the first few hours after chlorpyrifos treatment. In contrast to the effects seen after administration on PN1–4, shifting the period of chlorpyrifos exposure to PN11–14 had a much less notable effect, even when higher doses were used: no decreases in locomotor activity and overall increases in rearing and grooming that were not significantly gender-selective. Administration on PN11–14 did not produce differential effects on cholinesterase in males and females. These studies indicate that chlorpyrifos given during a critical neonatal period, even at levels below the threshold for overt toxicity, can elicit both immediate and delayed gender-selective behavioral abnormalities. The ultimate evaluation of the developmental neurotoxicity of chlorpyrifos will thus require long-term assessments of neurobehavioral consequences of exposure during discrete developmental periods.

Introduction

Chlorpyrifos (CPF) is heavily used for agricultural and domestic purposes due to its persistence and relative safety compared to other organophosphate insecticides. Unlike parathion, CPF evokes delayed neuropathies only with very high exposures [28], [42]. Nevertheless, there is increasing concern about exposure of pregnant women, infants and children, who may be exposed at or above the No Observable Adverse Effect Level [14], [16]. Indeed, some estimates indicate potential neonatal absorption of up to several mg/kg over a period of a few days after routine use [16]. Animal studies confirm that CPF has higher systemic toxicity in neonates, with over an order of magnitude lower LD50 values in neonates than in adult rats [37], [39], [54]. Perhaps more alarmingly, recent studies in rats have shown that CPF, in doses below the threshold for symptoms of systemic toxicity nevertheless can compromise the basic cellular processes of brain development (review [48]). These include inhibition of DNA synthesis [10], [51], [54], impaired acquisition of new cells [4], [44], reductions in macromolecules mediating cell differentiation [4], [21], slowed axonogenesis [11], [26], [51], altered functioning of neurotrophic signaling cascades [50], interference with gene transcription [8] and aberrant development of synaptic activity [9].

In light of the multiple neurochemical alterations produced by exposure of the immature brain to chlorpyrifos, one major question that needs to be addressed is how these cellular effects influence the development of behavioral performance. Although numerous studies have examined behavioral responses after adult or preweanling chlorpyrifos exposure [3], [5], [29], [31], [36], [38], or with maternal treatment during gestation [7], [32], the early neonatal period has received much less attention, despite the fact that, at this particular developmental stage, exposures are likely to be among the highest [16]. In the neonate, the adverse effects of cholinesterase inhibition are exacerbated by poor synaptic adaptability to cholinergic hyperstimulation [5], [36], and programming of brain cell reactivity and synaptic connections are at their peak and are thus most vulnerable [9], [48], [50]. The need to evaluate early childhood exposures has recently been reinforced by the observation that children exposed to domestic and agricultural pesticides who have never displayed any overt signs of intoxication, nevertheless perform significantly more poorly on neurobehavioral performance tests than do children in the same community, with the same ethnic and socioeconomic backgrounds, who have not been exposed [15].

In the current study, we administered CPF to neonatal rats in doses that do not evoke any overt signs of toxicity and then evaluated behavioral performance during the period of exposure, as well as in the postweaning period, several weeks after the termination of exposure. The effects were then compared to administration of higher doses during a later developmental stage, outside the window for many of the adverse effects on brain cell development [48].

Section snippets

Materials and methods

All experiments were carried out in accordance with the declaration of Helsinki and with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health. Timed-pregnant Sprague–Dawley rats (Zivic Miller Laboratories, Allison Park, PA) were housed in breeding cages with a 12 h light–dark cycle and with free access to food and water. Pups from all litters were randomized on the day after birth and redistributed to dams with litter sizes of 10±1

Results

In keeping with previous findings [4], [10], [21], [48], [50], [54], neither administration of 1 mg/kg of chlorpyrifos from PN1–4, nor administration of 5 mg/kg from PN11–14, caused any signs of overt toxicity: no mortality, no growth impairment, normal times of appearance of ear detachment and eye opening, and no discernible effects on suckling or maternal caretaking (data not shown).

Despite the lack of systemic toxicity, chlorpyrifos exposure had profound effects on the development of

Discussion

The results of this study indicate that neonatal exposure to chlorpyrifos, even at doses that do not cause systemic toxicity or any overt signs of intoxication, nevertheless alters the development of coordination skills and locomotor activity. These effects involve a critical period early in postnatal life, as deficits were not obtained when older animals were exposed to even higher doses of CPF. The results with neonates thus stand in direct contrast to long-term deficits after adult

Acknowledgements

The authors thank Dr. Stephanie Padilla at the U.S. Environmental Protection Agency for assistance with the cholinesterase measurements.

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    Supported by USPHS ES10387 and ES10356, and by a STAR Fellowship from the U.S. Environmental Protection Agency.

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