Gene Expression Analysis on the Early Development of Pig Embryos Exposed to Malathion

Abstract

Malathion is a widely used pesticide and there is evidence that it could alter mammal’s germ and somatic cells, as well as cell lines. There are not enough studies showing how the nonacute malathion doses affect gene expression. This study analyzes gene expression alterations in pig morular embryos exposed in vitro, for 96 h, to several malathion concentrations after in vitro fertilization. cDNA libraries of isolated morular embryos were created and differential screenings performed to identify target genes. Seven clones were certainly identified. Genes related to mitochondrial metabolism as cytochrome c subunits I and III, nuclear genes such as major histocompatibility complex I (MHC I), and a hypothetical protein related with a splicing factor were the target of malathion’s deregulation effect. The widespread use of malathion as a pesticide should be regarded with reproductive implications and more detailed analysis would yield more about molecular mechanisms of malathion injury on embryo cells.

Malathion (O,O-dimethyl-S-(1,2-dicarbethoxyethyl) phosphorodithionate) is one of the most widely used organophosphate insecticides throughout the world (Pluth et al. 1998). It is used to control pests affecting agricultural crops, ornamentals, greenhouses, livestock, stored grains, forest, buildings, households, and gardens. So big was the popularity of malathion that in Jonhson et al. (1979) found malathion residues on food products for human consumption and residues in infant formulations estimated in 0.003 to 0.01 and 0.006 to 0.173 mg/kg, respectively (Johnson et al. 1979).

The main toxic effect of malathion in insects is the irreversible inhibition of acetylcholinesterase by phosphorylation of serine residues in its active center, which leads to the accumulation of acetylcholine (Blasiak et al. 1999). However, for mammals malathion is considered slightly toxic because of its rapid modification to nontoxic α-monoacids by carboxylesterases, enzymes that can hydrolyze malathion and its metabolites to non-toxic intermediates that can be easily eliminated (Blasiak et al. 1999; Dauterman and Main 1966). Nevertheless, malathion is an alkylating agent and therefore can produce genotoxic damage (Chen et al. 1981; Giri et al. 2002; Wiaderkiewicz, Walter, and Reimschussel 1986), it has also been reported to produce Hprt mutations preferentially at G:C base pairs in human T lymphocytes (Pluth et al. 1998). In vivo and in vitro studies showed that malathion can elicit chromosomal aberrations, sister-chromatide exchange (Blasiak et al. 1999), as well as an increase of micronuclei frequency (Windham et al. 1998).

Because malathion is still a widely used pesticide, there is concern about its possible role on the reproductive physiology of vertebrates. There is evidence that malathion may elicit alterations in germ and somatic cells in mice (Salvadori et al. 1988) as well as in Chinese hamster cell line V79 and human lymphoid cell lines (Chen et al. 1981).

In this study the pig was chosen as the mammal model because porcine physiology is very similar to that of humans considering the reproductive and endocrine systems (Petters 1994). Gene expression was analyzed in porcine morular embryos exposed to 200 μM malathion after in vitro fertilization. cDNA libraries of isolated morular embryos were generated and differential screenings performed to identify target genes.

MATERIALS AND METHODS

Recovery and Culture of Oocytes

Chemicals for in vitro cultures were purchased from Sigma Chemical (St. Louis, MO). Culture conditions were 39°C with 5% CO₂ in air, humidified atmosphere, and culture media were covered with light mineral oil.

Ovaries were collected from gilts at a local slaughterhouse and transported to the laboratory in less than 1 hour. In vitro maturation (IVM) was performed as reported (Abeydeera et al. 1998; Betancourt, Fierro, and Ambriz 1993; Wang and Niwa 1995).

In Vitro Fertilization (IVF)

After IVM, cumulus cells were removed by adding 0.1% (w/v) hyaluronidase (H-3506). For IVF 30 to 35 denuded oocytes were washed and placed in 50-μl drops of Tris-buffer medium (TBM) and kept in the incubator until spermatozoa were added for fertilization.

Semen sample was processed as reported by Abeydeera et al. (1998). Oocytes were inseminated with sperm concentration of 5 × 10⁵/ml and coincubated for 7 h.

Embryo Culture

After sperm-oocyte coincubation, putative embryos were washed 3 times in embryo culture medium (NCSU-23), and transferred to a Nunc 4-well multidish. In the first well, embryos were developed only in 500 μl of medium, whereas in the other wells, embryos were exposed to 100, 150 and 200 μM of malathion, on each well, respectively. After 96 h, cleavage rate and morula formation were evaluated under a stereomicroscope, morulae were selected for the following steps.

Embryo Collection and Cell Lysis

Embryos at the morular stage were selected and washed 3 times with phosphate-buffered saline (PBS) and harvested in the smallest volume possible (1 μl) of sterile PBS (Microlab, Mexico), immediately frozen, and stored at –70°C until total RNA was extracted.

Library Construction

Retrotranscription was performed as reported by Bonilla and Del Mazo (2003) using whole-embryos lysates to avoid RNA loss during isolation. Two embryos were lysed by rapid thawing and freezing steps immediately before the assay.

Poly(dA) tailing of the 3′ end of the first-strand cDNA was done by using 12 U of terminal deoxynucleotidyl transferase (Roche-Diagnostics, Basel, Switzerland) as reported.

The oligo(dA)-tailed first-strand cDNA was polymerase chain reaction (PCR) amplified using the dT-EcoR1 primer for 25 cycles as it was described (Bonilla and Del Mazo 2003). cDNA obtained from control and malathion-treated embryos was used as a probe and for library construction. Library construction was performed independently twice in Zap Express II (Stratagene, La Jolla, CA) according to manufacturer instructions.

Selection of cDNA Clones by Differential Screening

Approximately 10,000 plaques were screened on each assay at a 2000 plaque-forming units (pfu)/plate density. Plaques were transferred onto nitrocellulose membranes in duplicate. One membrane was incubated with the radioactive cDNA probe obtained from untreated control embryos cDNAs, whereas the other was incubated with the radioactive cDNA probe from malathion-treated embryos. Differentially expressed genes were detected by autoradiography, selected, and PCR amplified using the M13–20 primer and the BK Reverse primer. Second differential analysis was done by Southern blot analysis in duplicate using the same hybridization conditions. Only clearly different signals appearing in the second screenings were considered genuinely positive (Bonilla and Del Mazo 2003).

Nucleotide Sequencing and Analysis of Sequences Homologies

PCR products were sequenced using an ABI PRISM 3100-Avant Genetic Analyzer. Analysis of sequence homologies was done using the Basic Local Alignment Search Tool (BLAST) algorithm at the National Center for Bioinformatics (NCBI) site (http://www.ncbi.nml.nih.gov/blast).

Data Analysis

Embryo development experiments were repeated six times. Control morulae (not exposed to malathion), were normalized to 100%. Difference was considered significant when p < .05 using two-sided t test.

RESULTS

After 96 h of IVF, the effect of malathion on the viability and survival of morular embryos was determined and healthy morulae were recovered (Figure 1). Healthy morulae were those composed of homogeneous and not vacuolated blastomers. Morula viability was assessed using 3-(4,5-dimethyl-2-thiazyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT). A 100% value was assigned to the average of morulae obtained on experiments where zygotes were not exposed to malathion. Zygotes exposed to 100 μM of malathion yield 113% morulae, then as the concentration of malathion increased to 150 and 200 μM, the yield of morulae declined to 69% and 50%, respectively (Figure 2). Therefore the mean inhibition concentration (IC₅₀) was set at 200 μM. Embryos exposed to IC₅₀ were selected for gene expression analysis because the number of morulae obtained decreased to the 50% with normal morphology.

cDNA libraries were generated using only two or three representative embryos. Amplification was similar in both cases (Figure 3). Analysis of the cDNA libraries obtained showed a titer of 10⁶ pfu/ml with 93% of recombinants, which were amplified to 10⁹ pfu/ml. Electrophoretic analysis and sequence of clones selected at random revealed no redundancy (Figure 4A).

To identify differentially expressed genes in morulae exposed to malathion, differential screenings were performed using cDNA of untreated embryos to compare against those of treated embryos (exposed 96 h to 200 μM malathion). Clones corresponding to deregulated genes, detected by differential autoradiography signals in the first screening, were subjected to a second differential screening using Southern blots for PCR-amplified cDNA (Figure 4). As shown in Table 1, nine genes were consistently found to be differentially expressed; all of them were down-regulated in morulae exposed to malathion. Two mitochondrial-encoded genes were identified: COX I (cytochrome oxidase I) and COX III (cytochrome oxidase III); two mitochondrial no-coding sequences were also found. Two nuclear encoded genes were identified as major histocompatibility complex I (MHC I), and hypothetical protein CBG01613. Three down-regulated genes did not match to any reported sequences on the Gene Data Bank (Table 1).

DISCUSSION

This study analyzed the gene expression alterations in porcine morular embryos exposed to malathion after IVF. The concentrations of malathion used in this study are comparable to those found in blood following various nonlethal human exposures to pesticides (Blasiak et al. 1999). According to our results the yield of morulae was slightly higher at concentration of 100 μM, but without significant difference. Nevertheless, there was a decrease in the production of morulae with higher concentrations (150 and 200 μM). This dose-response phenomenon has been previously reported (Johnson et al. 2002; Rodgers and Xiong 1997) and, although it refers to a response in the immune system, it is possible that low doses of malathion as an stressor could activate mechanisms that may accelerate cell metabolism, but more experiments should be done to validate the significance of this tendency.

Because of scarcity of material, RNA extraction was avoided and direct amplification after lysis was chosen. Two embryos were enough to construct representative PCR-based cDNA libraries similarly to a previous study (Bonilla and Del Mazo 2003). cDNA libraries showed a titer of 10⁶ with 96% of recombinants, which were amplified to 10⁹ pfu/ml.

The differential hybridization technique used allowed the identification of nine down-regulated genes in early embryos exposed to malathion. Malathion exposure caused down-regulation of two genes involved in the mitochondria respiratory chain (MRC), cytochrome c oxidase subunits I and III. Since 1977, malathion was suggested to behave as an inhibitor of mitochondrial electron transport system because it may be related to a possible change in the permeability of the inner mitochondrial membrane (Spetale, Morisoli, and Rodríguez-Garay 1977). Early mammalian embryos are initially adapted for glycolytic bioenergy production oxygen utilization increases during blastomer compaction at the eight-cell stage, and then again during blastocyst formation, in concert with increased rates of glucose utilization and lactate production (Knudsen and Green 2004). Therefore, the regulation and function of mitochondria in the embryo has implications for development because mitochondrial dysfunction may lead to impaired oxidative phosphorylation, overproduction of reactive oxygen species, and programmed cell death. Malathion could then play an important role on development and/or implantation failure, because it has been reported that mRNA transcripts implicated on mitochondrial biogenesis such as COXI and nuclear respiratory factor 1 (NRF1) have an influence on developmental capacity of embryos specially from morular stage onwards (May-Panloup et al. 2005). Moreover, studies on fibroblasts show that following exposure to malathion there is an induction of caspase-dependent apoptosis. Activation of caspases is the major signal to initiate apoptosis and caspase 9 activation follows the mitochondrial stress, which could be the result of the lipophilic nature of malathion and its interaction with the inner membrane, possibly affecting electron delivery and signal apoptosis as a result of mitochondrial injury (Masoud et al. 2003).

The role of the major histocompatibility complex class I (MHC I), is crucial during embryonic life, because mammalian embryos are potentially at risk from maternal immune attack (Warner et al. 2002). There is evidence that an overexpression of MHC I is crucial for fetus implantation because some fetus cells are destined to cross to the maternal side of placenta where they fuse with maternal cells (Brainbridge, Sargent, and Ellis 2001). Nevertheless, these studies have been made on trophoblast cells, indicating a blastocyst stage; there is no reference of the expression of human leukocyte antigen (HLA) on earlier embryos. Success for embryo implantation could be endangered when embryos in early stages of development are exposed to malathion because it alters the expression of MHC I at morular stages, but further studies are necessary to confirm this result.

Malathion seems also to down-regulate the expression of a gene with homology with a hypothetical protein CBG01613, a member of splicing factor P54 family (Shav-Tal and Zipori 2002). The expression of this protein has not been reported before in pig.

Until now, there are no report on the effects of malathion on mammalian embryos, nevertheless, it has been hypothesized that cholinesterase (ChEs) may be possibly involved in the development of non-nervous tissues and cells as well, starting from gametes (Falugi et al. 1991). Sea urchin zygotes exposed to diazinon presented changes in intracellular Ca²⁺ content, due to acetyl cholinesterase (AChE) inhibition of muscarinic receptors (Angelini et al. 2004). Also, there may be a link between organophosphates and endocrine disruption (Aluigi et al. 2005). Recently, a study on mammary epithelial cells show that, after an exposure of 24 h to malathion, there was an increased expression of two aldoketo reductases (AKR1C1 and AKR1C2) and one estrogen-responsive gene (EBBP) (Gwinn et al. 2005).

According to our results, the main malathion targets were mitochondrial genes and some nuclear genes not reported before to be affected by this organophosphate. Possibly, the lipophilic nature of malathion allowed it to reach the mitochondrial DNA and, as an alkylating agent, it could alter the gene regulation. Besides, it has been suggested that malathion exerts its cytotoxic effects via mitochondria, because mitochondrial membrane potential (ΔΨ _m ) disruption, intracellular Ca₂ ⁺ elevation, generation of reactive oxygen species (ROS) and ATP depletion is seen when cells are exposed to malathion (Chen et al. 2006). Silenced expression of COX subunits lead to mitochondrial dysfunction, which cause apoptosis of the blastocyst stage embryos (Cui et al. 2006). Then, it is possible that malathion could provoke a deleterious effect on early embryo development and implantation success by affecting expression of mitochondrial genes. However, the effects of malathion are not limited to the mitochondria. As seen in this study, malathion also modify the expression of nuclear genes as MHC I.

Attention should be paid to the widespread use of malathion as a pesticide because of its possible reproductive effects such as failed implantation and on development of mammalian embryos. Detailed analysis would yield more about molecular mechanisms of malathion injury on mammalian embryo cells.