Characterization of chlorpyrifos-induced apoptosis in placental cells

Abstract

The mechanism by which chlorpyrifos exerts its toxicity in fetal and perinatal animals has yet to be elucidated. Since the placenta is responsible for transport of nutrients and is a major supplier hormone to the fetus, exposure to xenobiotics that alter the function or viability of placenta cells could ostensibly alter the development of the fetus. In this study, JAR cells were used to determine if CPF and the metabolites 3,5,6-trichloro-2-pyridinol (TCP) and chlorpyrifos-oxon (CPO) are toxic to the placenta. Our results indicate that chlorpyrifos (CPF), and its metabolite chlorpyrifos-oxon (CPO) caused a dose-dependent reduction in cellular viability with CPF being more toxic than its metabolites. Chlorpyrifos-induced toxicity was characterized by the loss of mitochondrial potential, the appearance of nuclear condensation and fragmentation, down-regulation of Bcl-2 as well as up-regulation of TNFα and FAS mRNA. Pharmacological inhibition of FAS, nicotinic and TNF-α receptors did not attenuate CPF-induced toxicity. Atropine exhibited minimal ability to reverse toxicity. Furthermore, signal transduction inhibitors PD98059, SP600125, LY294002 and U0126 failed to attenuate toxicity; however, SB202190 (inhibitor of p38α and p38ß MAPK) sensitized cells to CPF-induced toxicity. Pan-caspase inhibitor Q-VD-OPh produced a slight but significant reversal of CPF-induced toxicity indicating that the major caspase pathways are not integral to CPF-induced toxicity. Taken collectively, these results suggest that chlorpyrifos induces apoptosis in placental cells through pathways not dependent on FAS/TNF signaling, activation of caspases or inhibition of cholinesterase. In addition, our data further indicates that activation of p38 MAPK is integral to the protection cells against CPF-induced injury.

Introduction

Chlorpyrifos (CPF), an ogranophosphate, is currently utilized as a pesticide on more than 40 crops food crops including such as peaches, citrus, almonds and grapes. CPF and its metabolites have been detected in farm animals, such as cattle, hogs and sheep (Ivey, 1979, Ivey and Palmer, 1979, Ivey and Palmer, 1981). Moreover, chlorpyrifos residues or its metabolites have been discovered in the diet of preschoolers (Fenske et al., 2002a), in the urine of children living in proximity to orchards (Lu et al., 2000, Fenske et al., 2002b), as well as in the cord blood of infants born to minority women living in urban settings (Whyatt et al., 2004).

The ubiquitous presence of chlorpyrifos residues and pesticide mixtures has raised concerns about the safety limits of these compounds. Although, CPF has been shown to be relatively safe in adult animals, newly discovered evidence indicates that CPF is a developmental neurotoxicant in the fetus and is thus harmful (Garcia et al., 2003). In animals and cellular models, chlorpyrifos inhibits neural cellular replication (Qiao et al., 2001), interferes with cellular differentiation (Crumpton et al., 2000), evokes oxidative stress, alters neurotransmission (Dam et al., 1999, Bloomquist et al., 2002, Karanth et al., 2006, Slotkin and Seidler, 2007) and induces neurobehavioral changes (Ricceri et al., 2006). Additionally, animals exposed to CPF in utero or as juveniles display motor and cognitive delays (Moser, 2000). In humans, elevated levels of chlorpyrifos in umbilical cord plasma are inversely associated with birth weight and length in children born to minority women (Whyatt et al., 2004). The literature indicates that chronic CPF exposure is associated with decreased birth weight and length. In addition, lower birth weights have specifically been documented among African Americans infants (Rauh et al., 2006, Perera et al., 2003, Perera et al., 2005) exposed to CPF in utero. Finally CPF exposure is associated with alterations in developmental and psychomotor indices in Mexican-American children (Eskenazi et al., 2007) and with immunological abnormalities (Thrasher et al., 2002). Consequently, chlorpyrifos is ranked 125 on 2001 CERCLA List and the United States Environmental Protection Agency (USEPA) has placed restrictive guidelines on its utilization specifically inside the home; however, despite these restrictions CPF still lingers in many dwellings (Whyatt et al., 2007).

Despite the mounting evidence that CPF can be harmful to children and the developing fetus, the mechanism(s) of action have yet to be fully elucidated. Although most studies have focused on the direct effect of CPF on the fetus, very few have assessed the effect of CPF on the placenta. This is disconcerting due to the fact that the placenta is a major supplier of nutrients, cytokines and hormones, which regulate implantation and fetal maturation. In addition, the placenta has been shown to serve as a de facto liver for the fetus and is capable of detoxifying xenobiotics through the actions of phase 1 and phase 2 metabolizing enzymes such as cytochrome P₄₅₀ isoenzymes, N-acetyltransferase, and UDP-glucuronosyl transferase. Hence, the placenta represents a possible model for predicting the effects of overall fetal development and viability once exposed to a xenobiotic.

Recent literature indicates that low-level exposure to environmental contaminants may indeed interfere with placental function (Myllynen et al., 2005, Sagik et al., 2007). For example, endosulfan inhibits aromatase activity; whereas, methomyl, pirimicarb, propamocarb, iprodion, lindane and bisphenol-A enhance placental aromatase activity (Nativelle-Serpentini et al., 2003). TCDD exposure is associated with fetus loss and the alteration of the secretion of chorionic gonadotropin hormone in primates (Guo et al., 1999, Chen et al., 2003, Myllynen et al., 2005). With regards to chlorpyrifos, CPF and/or its metabolites have been detected in the fetuses of dams administered CPF perinatally (Mattsson et al., 2000, Abdel-Rahman et al., 2002). Abdel-Rahman et al. (2002) further concluded that though the placenta presents a barrier of protection against CPF; at high doses, CPF and its metabolites can cross the placenta and enter into the fetus despite extensive maternal hepatic metabolism, as well as, distribute to all fetal tissues and plasma with elimination occurring at a slow rate. In addition, Souza et al. (2004) demonstrated that CPF alters the enzymatic activity of placental PI4-kinase and phosphatidylinositol handling suggesting that CPF may have membrane-disrupting properties. Given that exposure to xenobiotics can alter the function and viability of placenta cells, thereby ostensibly, altering the growth and development of the fetus, we examined whether chlorpyrifos and its metabolites are toxic to placental cells.