Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

The acute effects of organophosphorus intoxication are generally characterized by the irreversible inhibition of acetylcholinesterase (EC 3.1.1.7), resulting in a large excess of acetylcholine at cholinergic receptors. If untreated, these effects culminate in death by respiratory failure (1). The time of death after a single acute exposure may range from less than 5 min to nearly 24 h, depending upon the dose and route of exposure (2). Although there are many documented instances of organophosphorus intoxication in humans (3), the most widely publicized exposures occurred in 1994 when 12 liters of sarin (methylphosphonofluoridic acid 1-methylethyl ester) were released by terrorists in Matsumoto City, Japan, injuring 471 persons (7). The effective alleviation of postexposure symptoms by the administration of carboxylesterases (EC 3.1.1.1) as pretreatment drugs has become a focus of study (8).

Carboxylesterases are involved in the detoxification of organophosphorus compounds by two mechanisms: inactivation of these compounds by hydrolysis of ester bonds, or irreversible binding of the compounds at the carboxylesterase active site (11). The result of both of these mechanisms is the effective alleviation of acetylcholinesterase inhibition by reducing the concentration of free compound available for binding.

Carboxylesterases are a large family of broad-specificity esterases that are widely distributed in various tissues and appear to serve a broad range of physiologic functions. These enzymes hydrolyze fatty acyl esters such as palmitoyl-CoA; acylcarnitines; and mono-, di-, and triacylglycerols; and may participate in the transport of fatty acids across the endoplasmic reticulum or in the maintenance of membrane structure (12, 13). It has also been proposed that carboxylesterases play a primary role in xenobiotic metabolism and in the detoxification of organophosphorus agents present in the environment (14). They hydrolyze xenobiotics containing ester, thioester, or amide groups and thus play a major role in the biotransformation of foreign chemicals, including many insecticides and drugs (18). According to the classification of Aldridge, carboxylesterases, which are inhibited by organophosphorus compounds, are called "B" esterases (19). Several members of this family have been characterized at the molecular level in tissues other than lung (20).

Although inhalation is a major exposure route of organophosphorus compounds, research to date on pulmonary carboxylesterases has been limited. Gaustad and colleagues (26) reported the purification and characterization of a carboxylesterase from rat lung that is inhibited by the organophosphorus compounds soman (methylphosphonofluoridic acid 1,2,2-trimethylpropyl ester) and sarin. However, low yields (1.0 mg protein/70 g lung tissue) prevented thorough characterization of this enzyme (26). Munger and associates have described a serine esterase released by human alveolar macrophages that is closely related to liver microsomal carboxylesterase (27). They also proposed a role for this enzyme in the detoxification of harmful xenobiotics. The inability to clone a full-length complementary DNA (cDNA) from alveolar macrophage RNA limited the molecular characterization of the enzyme.

In view of the potential role of lung carboxylesterase in the detoxification of inhaled organophosphorus agents, we report the cloning, sequencing, and expression of the first full-length lung carboxylesterase. The localization of this lung carboxylesterase to airway epithelium and its inhibition by paraoxon (phosphoric acid diethyl 4-nitrophenyl ester) indicate that it may play a role in scavenging inhaled organophosphorus compounds.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

RNA Isolation and Reverse Transcriptase Polymerase Chain Reaction

Total RNA was prepared from freshly isolated male Sprague-Dawley rat lung, using TriReagent (Molecular Research Center, Inc., Cincinnati, OH) or guanidinium isothiocyanate as described by Chirgwin and coworkers (28). First-strand cDNA was synthesized from 10 µg of total RNA from rat lung and oligo dT as a primer using the Superscript Preamplification System (GIBCO BRL, Gaithersburg, MD) according to the manufacturer's instructions. A sense polymerase chain reaction (PCR) primer, XHO (5'-CTCGAGCTGTGGGCTAT T TGTTC-CTTCCAC), corresponding to bases 25 to 2 of the carboxylesterase, hepatic cholesteryl ester hydrolase (CEH), cDNA with an XhoI site (underlined) on the 5' end; and an antisense primer, XBA (5'-GCTCTAGAAGCAATAAGGTTAAGTATTTTCCC), corresponding to bases 1828 to 1851 of the hepatic CEH cDNA with an XbaI site (underlined) on the 5' end, were synthesized. A search of the GenEMBL database revealed that no matches for either primer were detected that would interfere with their intended use in PCR. Fifteen percent of the first-strand product was used to amplify the target cDNA. The PCR mixture composed of 1× PCR buffer (20 mM Tris-HCl, pH 8.4/50 mM KCl), 1 mM dithiothreitol, 0.2 mM deoxynucleotide triphosphates and 20 pmol of the hepatic-specific primers in a final volume of 100 µl was used. A total of 1.5 mM MgCl₂ was added separately to the reaction mix and overlaid with mineral oil, then amplified with the Coy TempCycler II, model 110S (Ann Arbor, MI). Taq DNA polymerase, 2.5 U, was added after an initial 5-min cycle at 94°C. The amplification profile consisted of 25 cycles of denaturation at 94°C for 30 s, primer annealing at 55°C for 45 s, and extension at 72°C for 2 min, with a final extension at 72°C for 10 min.

Cloning and Sequencing of Lung Carboxylesterase cDNA

A total of 42 ng of PCR product was used to ligate with 60 ng of pCR3 vector using the eukaryotic TA cloning kit (Invitrogen, San Diego, CA). The ligated plasmid was used to transform TOP10F' cells, and recombinant plasmid DNA was prepared from individual colonies. Sequence information from both ends was obtained using plasmid-specific T7 primer that reads through the 5' PCR primer XHO and SP6 primer that reads through the 3' PCR primer XBA to identify plasmid with correct orientation. Lung cDNA was sequenced by "oligo walking" using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer, Foster City, CA). Reaction mixtures, 20 µl, were made with 450 ng of the eukaryotic expression vector pCR3 containing the correct insert, 8.0 µl of the terminator ready reaction mix (A-Dye Terminator, C-Dye Terminator, G-Dye Terminator, T-Dye Terminator, dITP, dATP, dCTP, dTTP, Tris-HCl [pH 9.0], MgCl₂, thermal stable pyrophosphatase, and AmpliTaq DNA Polymerase, FS), and 3.2 pmol of either sense or antisense primers. Each reaction mixture was overlaid with one drop of mineral oil. The cycle sequencing profile consisted of denaturation at 96°C for 15 s, primer annealing at 50°C for 7 s, and extension at 60°C for 4 min, repeated for 25 cycles. Centri-Sep spin columns (Princeton Separations, Inc., Adelphia, NJ) were used according to the manufacturer's instructions to remove excess DyeDeoxy terminators from the completed DNA sequencing reactions. The reactions were analyzed on the Applied Biosystems DNA Sequencer 373A (Foster City, CA). The sequence obtained was mapped for restriction endonucleases using the GCG program.

Confirmation of cDNA by Alternative Cloning

The previous experiment was repeated from a separate RNA preparation, and a second full-length clone was obtained and sequenced as described. The full-length clone was analyzed by the restriction enzymes BamHI, which yields a 500-base pair (bp) fragment, and HindIII, which yields a 1,500-bp fragment, to confirm the correct sense orientation of the insert. Further, using 5' rapid amplification of cDNA ends (RACE) and reverse transcriptase (RT)-PCR, three additional clones were obtained spanning the same region of cDNA that was sequenced from the first clone. The primers synthesized for this additional cloning, and the corresponding bases of the lung carboxylesterase cDNA with which they match, are as follows (Figure 1):

View larger version (82K): [in this window] [in a new window]   Figure 1.   The cDNA nucleotide sequence and the deduced amino acid sequence of rat lung carboxylesterase. The first nucleotide of the full-length cDNA and the methionine residue translating the start codon are numbered as 1. The conserved active site motif (GXSXG) is shown in boldface and underlined. Asterisks indicate potential N-glycosylation sites. The 19 amino acids corresponding to the lung carboxylesterase previously purified (17) are underlined.

Antisense primers. ASP3 (5'-TCAGAGATTTTCAGTGTTGG), corresponding to bases 1153-1172; ASP4 (5'-CCAGGAGTTCATCCTCTGTC), corresponding to bases 864-883; and ASP1 (5'-CCAGTGGGATGGTTCCTCTG), corresponding to bases 1661-1680.

Sense primer. SP2 (5'-TTCAGCACAGGGGATGA), corresponding to bases 532-548.

Eukaryotic Expression System

COS-7 cells were transfected with the plasmid pCR3 containing the full-length cDNA described previously in CLONING AND SEQUENCING OF LUNG CARBOXYLESTERASE CDNA, using lipofectamine (GIBCO BRL) according to the manufacturer's instructions. Control cells were mock-transfected with lipofectamine alone. After 48 h of incubation the cells were harvested in homogenizing buffer containing 250 mM sodium phosphate, pH 7.4, and 250 mM sucrose. The cell suspension was sonicated using a Heat Systems Ultrasonic Processor (Farmingdale, NY), and the cell debris was removed by centrifugation at 10,000 × g for 30 min at 4°C. The postmitochondrial supernatant was assayed for carboxylesterase activity as described below.

Enzyme Assays and Protein Estimation

To measure activity toward p-nitrophenyl acetate (Sigma Chemical Co., St. Louis, MO), an aliquot of the supernatant was diluted with 200 mM phosphate buffer, 5 mM -mercaptoethanol, and 80 mM potassium chloride, in a final volume of 1 ml. The reaction was started by the addition of p-nitrophenyl acetate or p-nitrophenyl caprylate to a final concentration of 0.2 mM and incubated at 37°C for 5 min. The release of p-nitrophenol was monitored at 400 nm. Protein was estimated by Pierce BCA reagent (Pierce, Rockford, IL) using bovine serum albumin as a standard.

Northern Blot Analysis

Total rat lung RNA, 10 µg, was electrophoresed on a 0.7% agarose gel in the presence of formaldehyde. The RNA was stained with ethidium bromide and the integrity of the isolated RNA was verified by the presence of 28S and 18S ribosomal RNA bands. The RNA was transferred to a GENESCREEN membrane (NEN Research Products, Boston, MA) and hybridized with a nick-translated, ³²P- labeled, full-length lung carboxylesterase cDNA according to the manufacturer's instructions. The blots were washed under low stringencies (2× saline sodium citrate [SSC] + 0.1% sodium dodecyl sulfate [SDS] at room temperature) and high stringencies (0.2× SSC + 0.1% SDS at 60°C). Positive hybridization was detected by exposure to Kodak XAR-2 films for 16 h at 70°C.

In Situ Hybridization

Under pentobarbitol anesthesia, rats were transcardially perfused with heparinized phosphate-buffered saline (PBS) (100 mM sodium phosphate, 150 mM NaCl [pH 7.2], and PBS) followed by fixative consisting of 4% paraformaldehyde and 0.1% glutaraldehyde. The lungs were removed and fixed for an additional 24 h with the same fixative, dehydrated, and embedded in paraffin wax. Ten-micron sections of the lower lobe of the right lung were processed as follows (29):

Paraffin sections were dewaxed in xylene and hydrated through graded ethanol concentrations to PBS. Permeabilization was carried out for 2 min in 0.01% Triton X-100 in PBS. Sections were then treated with proteinase K (1 µg/ ml in 20 mM Tris-HCl/5 mM ethylenediaminetetraacetic acid) for 30 min at 37°C. The reaction was stopped by postfixation in buffered 4% paraformaldehyde for 15 min, followed by rinsing in PBS for 5 min. Sections were then acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine for 10 min, dehydrated through graded ethanol solutions, and air-dried.

The hybridization buffer contained 50% formamide, 4× SSC, 1× Denhardt's solution, 10% dextran sulfate, 0.24 µg/µl yeast RNA, 0.5 µg/µl salmon sperm DNA, 1% sarcosyl, 2.4 mg/ml Na₂PO₄, and 1 pmol/ml of the biotin-labeled probe that is complimentary to the 5' PCR primer used to clone the rat lung carboxylesterase described above. Hybridization buffer, 500 µl, was applied to each slide and covered with a PROBE-CLIP (Grace Bio-Labs, Sunriver, OR) to prevent evaporation. Hybridization was carried out overnight in moist chambers at 37°C. After hybridization, the sections were washed four times in 0.5× SSC for 15 min at room temperature. Sections were then incubated at room temperature for 1 h in blocking solution (PBS containing 1% normal goat serum, 0.1% Triton X-100, and 5% nonfat dry milk solids). Sections were then incubated with rhodamine conjugated to streptavidin for 1 h at room temperature. After three 30-min washes with PBS, slides were air-dried and mounted in glycerol/N-propyl galate. Affinity labeling was visualized using the Meridian Instruments Ultima (Okemos, MI) scanning confocal fluorescence microscope with 516 nm laser excitation and dual-wavelength detection of emission.

Inhibition of Lung Carboxylesterase by Organophosphorus Compounds

The full-length lung carboxylesterase cDNA was transfected into COS-7 cells as described previously and 0.1, 1.0, and 10.0 µM paraoxon were incubated with between 35 and 45 µg of protein for 15 and 30 min at 37°C. To measure the decrease in activity toward p-nitrophenyl caprylate (Sigma), an aliquot of the supernatant was diluted with 200 mM phosphate buffer, 5 mM -mercaptoethanol, and 80 mM potassium chloride in a final volume of 1 ml. The reaction was started by the addition of p-nitrophenyl caprylate to a final concentration of 0.2 mM and incubated at 37°C for 15 min. The release of p-nitrophenol was monitored at 400 nm.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Cloning the Rat Lung Carboxylesterase cDNA

RT-PCR was used to amplify the cDNA of rat lung carboxylesterase from total rat lung RNA. The cDNA was amplified and cloned into the TA cloning vector pCR3. Digestion of the plasmid DNA with EcoRI showed that this clone contained an insert of approximately 2 kb. The cDNA sequence and predicted amino acid sequence obtained from this clone are shown in Figure 2. The first ATG codon, 11 bp from the 5' terminus, was identified as the start codon by the sequence CCACAATG, differing only in the fifth nucleotide from the consensus sequence, CCACCATG, described by Minchiotti and colleagues (30). The nucleotide sequence surrounding this ATG codon also corresponds to the optimal consensus sequence AXXATGXG for the initiation of translation by eukaryotic ribosomes, as described by Kozak (31). Beginning with the consensus initiation codon, there exists a long open reading frame coding for 565 amino acids. A termination codon, TGA, is present at 1,696 bp, leaving a 143-bp 3' untranslated region. The predicted molecular weight of the nonglycosylated protein is 62,117, and there are two potential N-glycosylation sites at Asn⁷⁹ and Asn⁴⁸⁹.

View larger version (7K): [in this window] [in a new window]   Figure 2.   The cloning strategy used to determine the cDNA sequence of rat lung carboxylesterase. Primers XHO and XBA produced two full-length 1,839-bp clones. UAP and ASP4, SP2 and XBA, and SP2 and ASP1 produced three separate clones that were sequenced to confirm the cDNA rat lung carboxylesterase sequence, as described in MATERIALS AND METHODS.

Verification of the Lung Carboxylesterase

As shown in Figure 1, two full-length clones were obtained by RT-PCR and sequenced in the forward and reverse directions. To confirm this sequence, three additional clones were obtained as described in MATERIALS AND METHODS, and each was sequenced in both the forward and reverse directions. This thorough cloning method eliminated the possibility of sequencing errors. The authenticity of the 1,839-bp lung carboxylesterase was further verified by expression in COS-7 cells transfected with the pCR3 plasmid containing the full-length cDNA. Expression was driven by the cytomegalovirus promoter located upstream of the 5' end of the cDNA. As shown in Figure 3, a 4-fold higher activity toward p-nitrophenyl acetate and a 5.5-fold higher activity toward p-nitrophenyl caprylate was present in extracts from cells transfected with the lung carboxylesterase cDNA as compared with mock-transfected controls. Chinese hamster ovary cells were also used to express the 1,839-bp lung carboxylesterase to further verify its authenticity. A 3.2-fold higher activity toward p-nitrophenyl acetate was observed in extracts from cells transfected with the lung carboxylesterase cDNA when compared with controls treated with lipofectamine alone (data not shown).

View larger version (19K): [in this window] [in a new window]   Figure 3.   Lung carboxylesterase activity in transfected COS-7 cells. Panel A shows activity toward p-nitrophenyl acetate and panel B shows activity toward p-nitrophenyl caprylate. The cells were transfected with lipofectamine alone (squares) or with lipofectamine and pCR3 containing the carboxylesterase cDNA (circles and triangles). Cells were harvested and lysates assayed for carboxylesterase activity as described in MATERIALS AND METHODS. Activity is expressed as nanomoles of para-nitrophenol released/h.

Total RNA from rat lung was probed with the full-length lung carboxylesterase cDNA on Northern blots. As shown in Figure 4, the probe hybridized to a single 2.5-kb band, indicating the presence of the messenger RNA (mRNA) in the lung.

View larger version (13K): [in this window] [in a new window]   Figure 4.   Northern blot analysis of rat lung RNA probed with lung carboxylesterase cDNA. Total RNA (10 µg) from rat lung was electrophoresed on an agarose gel, transferred to membrane, hybridized to the radiolabeled full-length cDNA probe, and developed by autoradiography, as described in MATERIALS AND METHODS. The 28S and 18S RNA bands were visualized by ethidium bromide staining (not shown) and marked.

Cellular Localization of the Lung Carboxylesterase

Intratissue localization of the rat lung carboxylesterase mRNA was determined using the novel in situ hybridization technique. Lung tissue sections were hybridized overnight with a biotin-labeled probe, complementary to the 5' PCR primer used to clone the full-length lung carboxylesterase cDNA. Specific hybridization of this biotinylated probe was detected using rhodamine-conjugated streptavidin. Affinity labeling was visualized using the Meridian Instruments OPTIMA scanning confocal fluorescence microscope with 516 nm laser excitation and dual-wavelength detection of emission. In Figure 5, panel 2, the positive signal is the bright generalized glow, indicating the presence of the message in the epithelial lining of the small-diameter airway at the terminal bronchiole level. The surrounding smooth muscle and parenchyma also show the expression of the message, although at relatively lower levels. Also shown in Figure 5 is a serial lung section stained with hematoxylin and eosin to define more clearly the structure of the airway epithelium.

View larger version (51K): [in this window] [in a new window]   Figure 5.   In situ hybridization in rat lung tissue sections. A 10-µm sample section (panel 2) was hybridized overnight with biotin-labeled probe complimentary to the 5' PCR primer used to clone the rat lung carboxylesterase described in MATERIALS AND METHODS. The section was then incubated with rhodamine conjugated to streptavidin. Affinity labeling was imaged using the Meridian Instruments OPTIMA scanning confocal fluorescence microscope with 516 nm laser excitation and dual-wavelength detection of emission. A 10-µm serial control sample (panel 1) was treated identically to the sample section, except for hybridization with probe. A 10-µm serial section was stained with hematoxylin and eosin (panel 3), and the boxed area represents the area shown in panels 1 and 2. A ×60 objective was used in panels 1 and 2, and a ×20 objective was used in panel 3.

Organophosphorus Inhibition of the Lung Carboxylesterase

The inhibitory effect of the organophosphorus compound paraoxon toward the lung carboxylesterase was also determined. The amounts of 0.1, 1.0, and 10 µM paraoxon were incubated with the transfected lung carboxylesterase for 15 and 30 min and the reaction was started by the addition of p-nitrophenyl caprylate. The activity of the transfected protein was assayed as described previously. The incubation with 0.1 µM paraoxon inhibited 93% of the transfected carboxylesterase activity after incubation with 40 µg of the protein for 15 min (Figure 6). After a 30-min incubation with 40 µg of the transfected carboxylesterase, 0.1 µM paraoxon inhibited 100% of the transfected carboxylesterase activity (Figure 6).

View larger version (15K): [in this window] [in a new window]   Figure 6.   Paraoxon inhibition of rat lung carboxylesterase. The full-length lung carboxylesterase cDNA was transfected into COS-7 cells as described in MATERIALS AND METHODS. Expressed protein (35 to 45 µg) was incubated with 0.1 µM paraoxon for 15 and 30 min. The inhibitory effect of the paraoxon toward the activity (nmoles/h) of the transfected lung carboxylesterase was determined by measuring the hydrolysis of p-nitrophenyl caprylate as described in MATERIALS AND METHODS. *P < 0.05.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Comparisons between the rat lung carboxylesterase and other published carboxylesterase sequences show that the lung carboxylesterase reported here is closely related to hepatic CEH (20), pI 6.1 esterase (21), and hydrolase A (22), all characterized from the liver. The amino acid sequence translated from the cDNA reported here (Figure 1) exhibits 100% identity with the first 19 N-terminal amino acids of the principal carboxylesterase in the rat lung purified by Gaustad and associates (26). In contrast to reported subcellular distribution of rat liver carboxylesterases, this rat lung carboxylesterase was shown to be predominantly in the cytosolic fraction, whereas negligible activity was present in the microsomes (26). Experiments in this laboratory also show 80% of activity toward p-nitrophenyl acetate in the cytosolic fraction versus only 20% in the microsomal fraction (data not shown). Therefore, the lung carboxylesterase cDNA reported here probably encodes the cytosolic carboxylesterase characterized by Gaustad and coworkers (26).

Carboxylesterases belong to the family of serine esterases, which are characterized by a conserved catalytic triad (serine, histidine, and an acidic residue). As shown in Table 1, domains associated with the catalytic triad are present in the lung carboxylesterase. These conserved regions contain putative active site residues, namely, Asp¹¹⁵ in a SEDCLY motif, Ser²²¹ in a GXSXG motif, Glu²⁴⁶ in an SES motif, and His⁴⁶⁶ in a GDHGD motif (23). The relationship between the lung carboxylesterase reported here and other carboxylesterases is further confirmed by expression of the active enzyme. As shown in Figure 3, extracts from COS-7 cells transfected with the lung carboxylesterase cDNA exhibited a 4-fold increase in activity toward p-nitrophenyl acetate and a 5.5-fold increase in activity toward p-nitrophenyl caprylate. Despite high homology with CEH, no CEH activity was present in extracts from COS-7 cells transfected with the lung carboxylesterase cDNA (data not shown).

Organophosphorus compounds are highly lipophilic liquids generally characterized by high vapor pressures. Used as insecticides or chemical weapons, they are often dispersed as aerosols that are rapidly absorbed by the lungs after inhalation (2). After inhalation, these compounds can readily be absorbed across the plasma membrane of the epithelial cells and detoxified by cytosolic carboxylesterases.

From a toxicologic standpoint, the tissue carboxylesterase that first encounters an organophosphorus compound is primarily responsible for its detoxification (32). Among all tissues that are permanently exposed to the environment, the lung provides the largest surface of primary contact with air contaminated with xenobiotic vapors, aerosols, and particles (33). Therefore, the pulmonary carboxylesterase may serve as a detoxifying enzyme against inhaled organophosphorus agents. The results from this study indicate the presence of a lung carboxylesterase in airway epithelium (Figure 5), where it would come in direct contact with and be able to inhibit the effects of inhaled organophosphorus agents.

It has been shown that organophosphorus compounds react irreversibly with carboxylesterases, and the resulting inhibition produces no physiologic toxicity (34). Therefore, carboxylesterases act as scavengers that reduce the amount of organophosphorus compound available to inhibit acetylcholinesterase. The detoxification of the more toxic organophosphorus agents such as soman is carried out primarily by the lung and plasma carboxylesterases. Moreover, when carboxylesterase activity is inhibited before organophosphorus poisoning, the lethality of the effects has been shown to be markedly potentiated (17). As shown in Figure 6, there is a total inhibition of the lung carboxylesterase by the organophosphorus agent paraoxon at a concentration of 0.1 µM after a 30-min incubation, suggesting that this enzyme may play a role in scavenging these toxic agents and thereby reducing their lethality.

This study describes the first full-length gene product encoding a lung carboxylesterase, which will facilitate thorough characterization of the role this enzyme plays in organophosphorus detoxification. Its localization to the epithelial surfaces of the airways and its complete inhibition by paraoxon (allowing tentative classification as a B esterase) indicate that it may play a role in the detoxification of inhaled organophosphorus agents.