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Vol. 29, Issue 9, 1201-1204, September 2001
Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina (J.T., Y.C., R.L.R., E.H.); United States Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, Maryland (A.A.B.); and National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina (D.D., J.A.G.)
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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One of the factors determining the toxicity of chlorpyrifos (CPS), an organophosphorus (OP) insecticide, is its biotransformation. CPS can be activated by cytochrome P450 (CYP) through a desulfuration reaction to form chlorpyrifos-oxon (CPO), a potent anticholinesterase. CPS can also be detoxified by CYP through a dearylation reaction. Using pooled human liver microsomes (HLM), a Kmapp of 30.2 µM and Vmaxapp of 0.4 nmol/min/mg of protein was obtained for desulfuration, and a Kmapp of 14.2 µM and a Vmaxapp of 0.7 nmol/min/mg of protein was obtained for dearylation. These activities are lower than those obtained from rat liver microsomes. Gender differences in humans were also observed with female HLM possessing greater activity than male HLM. Use of human CYP isoforms expressed in human lymphoblastoma cells demonstrated that CYP1A2, 2B6, 2C9*1, 2C19, and 3A4 are involved in CPS metabolism. CYP2B6 has the highest desulfuration activity, whereas dearylation activity is highest for 2C19. CYP3A4 has high activity for both dearylation and desulfuration. The use of phenotyped individual HLM demonstrated that predictions of metabolic activation and/or detoxication could be made based on relative amounts of CYP2B6, 2C19, and 3A4 in the microsomes. Thus, individuals with high CYP2C19 but low 3A4 and 2B6 are more active in dearylation than in desulfuration. Similarly, individuals possessing high levels of CYP2B6 and 3A4 have the greatest potential to form the activation product. These differences between individuals suggest that differential sensitivities to CPS may exist in the human population.
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Introduction |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Chlorpyrifos [O,O-diethyl-O-(3,5,6-trichloro-2-pyridinyl)-phosphorothioate] (CPS1) is a widely used organophosphorus (OP) insecticide. It has numerous agricultural applications and, until recently, has been used for termite control in foundations and for the control of nuisance insects and disease vectors in homes and during military deployments. The extensive use of CPS inevitably results in human exposure and has the potential to cause toxic effects.
The in vivo toxicity of CPS is a result of its bioactivation by
cytochrome P450 (CYP)-mediated monooxygenases to a more potent cholinesterase inhibitor, chlorpyrifos-oxon (CPO). This oxidation reaction, which proceeds through a possible phosphooxythiiran intermediate, can result in either a desulfuration reaction that generates the oxon or a dearylation reaction that degrades the parent
compound (Chambers, 1992
) (Fig. 1).
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Studies of parathion, a related organophosphate, have shown that
parathion oxidation is catalyzed by human CYP1A2, 2B6, and 3A4 and that
its oxidation is highly correlated to CYP3A4 activity in human liver
microsomes (HLM) (Butler and Murray, 1997; Mutch et al., 1999). Other
studies using specific chemical inhibitors for human CYP isoforms
demonstrated that both CYP2D6 and CYP3A4 were active in the
desulfuration of parathion, CPS, and diazinon (Sams et al., 2000).
The present study was designed to 1) determine oxidation activities toward CPS in human, mouse, and rat liver microsomes in the same assay system, 2) identify the human CYP isoforms and CYP polymorphic forms responsible for CPS oxidation, and 3) examine the differences in CPS oxidation activities among liver microsomes from selected individual humans.
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Chemicals. CPS, CPO, and 3,5,6-trichloro-2-pyridinol (TCP) were purchased from ChemService (West Chester, PA). HPLC grade acetonitrile and methanol were purchased from Fisher Scientific (Fair Lawn, NJ). All other chemicals, if not specified, were purchased from Sigma (St. Louis, MO).
Rodent Liver Microsome Preparation.
Rat liver microsomes (RLM) and mouse liver microsomes (MLM) were
prepared from adult male Long-Evans rats and adult male CD-1 mice
(Charles River Laboratories, Raleigh, NC), respectively, according to
the method of Cook and Hodgson (1983). Briefly, immediately after
sacrificing the animals, the fresh livers were removed, weighed,
minced, and then homogenized with a Polytron homogenizer (Brinkmann Instruments, Westbury, NY) in 50 mM potassium phosphate buffer (pH 7.5) containing 0.1 mM EDTA and 1.15% potassium chloride. The homogenate was centrifuged at 10,000g for 15 min. The
supernatant was filtered through glass wool and centrifuged at
100,000g for 1 h. The pellet was resuspended in 50 mM
potassium phosphate buffer (pH 7.5) containing 0.1 mM EDTA and 0.25 M
sucrose. All processes were performed at 0 to 4°C. The
microsomal preparation was aliquoted and stored at 
80°C until use.
Protein concentration was determined using a BCA kit (Pierce, Rockford, IL).
Human Liver Microsomes and Human Cytochrome P450 Isoforms.
Pooled HLM (pooled from 10 donors), individual HLM, and human
lymphoblast-expressed CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9*1
(Arg144, Ile359), 2C9*2
(Cys144), 2C19, 2D6*1 (2D6-Val), 2E1, 3A4, and 4A11 were purchased from GENTEST (Woburn, MA). Pooled male and pooled
female HLM (pooled from 10 male donors and 10 female donors, respectively) were purchased from XenoTech, LLC (Kansas City, KS).
Different mutant alleles of human CYP2C19 were expressed in
Escherichia coli, according to the method of Luo et
al. (1998). NADPH-CYP reductase was obtained from Oxford Biomedical
Sciences (Oxford, MI).
In Vitro Chlorpyrifos Metabolism. Enzyme kinetic assays for microsomes were performed by incubation of serial concentrations of CPS (final concentration range, 2-100 µM) with microsomes in 100 mM Tris-HCl buffer (pH 7.4 at 37°C) containing 5 mM MgCl2 and 3 mM EDTA for 5 min. The microsomal protein concentrations used in assays were 1.5 mg/ml for HLM, 0.5 mg/ml for RLM, and 1 mg/ml for MLM. After preincubation at 37°C for 3 min, reactions were started by the addition of an NADPH-generating system (0.25 mM NADP, 2.5 mM glucose 6-phosphate, and 2 U/ml glucose-6-phosphate dehydrogenase). The controls were identical except for the absence of an NADPH-generating system. Reactions were terminated by adding an equal volume of ice-cold methanol and vortexing. After 5 min of centrifugation at 15,000 rpm in a microcentrifuge, the supernatants were analyzed for CPO and TCP concentrations by HPLC.
Metabolic activity assays for human lymphoblast-expressed CYP isoforms were performed by incubation of CPS (final concentration, 100 µM) with CYP isoforms (final protein concentration, 0.9 mg/ml; final P450 contents, 23.4-180 pmol/ml) for 20 min in CYP-specific buffers recommended by the supplier (GENTEST). For CYP1A1, 1A2, 2D6, and 3A4, 100 mM potassium phosphate buffer with 3.3 mM MgCl2 (pH 7.4) was used. For CYP2B6, 2C8, 2C19, and 2E1, 50 mM potassium phosphate buffer with 3.3 mM MgCl2 (pH 7.4) was used. For CYP2C9*1, 2C9*2, and 4A11, the buffer was 100 mM Tris-HCl buffer with 3.3 mM MgCl2 (pH 7.5), whereas for CYP2A6, 50 mM Tris-HCl buffer with 3.3 mM MgCl2 (pH 7.4) was used. The metabolic activity assays for E. coli-expressed human CYP2C19s were performed according to the method of Klose et al. (1998). Briefly,
L-
-phosphatidylcholine (0.3 µg/pmol of
P450), CYP reductase (4 pmol/pmol of P450), and CYP2C19 (24 pmol) were combined and preincubated at 37°C for 5 min. This mixture then was
incubated with 100 µM CPS in 50 mM potassium phosphate buffer with
3.3 mM MgCl2 (pH 7.4) for 10 min. The reaction
was initiated with NADPH-generating system as described previously.
Assays of individual HLM (final protein concentration, 1.5 mg/ml) with
CPS (final concentration, 100 µM) were described previously.
Analysis of Metabolites by HPLC.
The HPLC system used in this study consisted of two Shimadzu (Kyoto,
Japan) pumps (LC-10AT), a Shimadzu auto injector (SIL-10AD VP), and a Waters 486 tunable absorbance detector (Milford,
MA). The mobile phase for pump A was 10% acetonitrile, 89% water, and 1% phosphoric acid, whereas that for pump B was 99% acetonitrile and
1% phosphoric acid. A gradient system was initiated at 20% pump B and
increased to 100% pump B in 20 min. The flow rate was 1 ml/min.
Metabolites were separated by a C18 column (Luna
5 µ, 150 × 3 mm; Phenomenex, Rancho Palos Verdes, CA) and
detected at 230 nm. Using this system, the retention times obtained for TCP, CPO, and CPS were 8.5, 12, and 17 min, respectively. The limits of
detection for TCP and CPO were 0.03 and 0.04 µM, respectively, at an
injection volume of 15 µl. Concentrations of metabolites were
obtained by extrapolation of peak height from a standard curve.
Kmapp and
Vmaxapp were obtained using a Hanes-Woolf
plot (Segel, 1975).
Statistics.
Significant differences between data sets were determined by one-way
analysis of variance, and multiple comparisons were performed with the
Tukey-Kramer method using an SAS program (SAS, 1989).
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Results |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The protein concentrations and incubation times used in the assays were within linear ranges determined in preliminary experiments. No metabolites were detected when incubations were carried out in the absence of an NADPH-generating system.
HLM displayed lower affinity (i.e., higher Kmapp) and lower reaction velocity toward CPS for both desulfuration and dearylation than RLM (Table 1). Compared with HLM, MLM exhibited similar affinities but a higher reaction velocity toward CPS (Table 1). Both RLM and MLM have higher values of clearance terms (Vmax/Km) than HLM. Pooled female HLM showed significantly higher metabolic activity toward CPS than pooled male HLM (Table 2).
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A screen of several human CYP isoforms demonstrated that CYP1A2, 2B6, 2C9*1, 2C19, and 3A4 were involved in CPS metabolism (Table 3), whereas no oxidation activity toward CPS was detected using CYP1A1, 2A6, 2C8, 2C9*2, 2D6, 2E1, and 4A11. Desulfuration and dearylation activities were greatest for CYP2B6 and CYP2C19, respectively. Marked decreases in metabolic activity toward CPS were observed with different polymorphic alleles of CYP2C19 (Table 4).
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To determine the variation range of CPS metabolism between individuals, we examined CPS metabolism from five individuals representing contrasting activities of some important CYP isoforms (Table 5). Individuals with high levels of CYP2B6 and 3A4 (HG042 and 112) had high-desulfuration activity; individuals with low levels of CYP2B6 and 3A4 (HG006, 023 and 043) had low-desulfuration activity. The dearylation pathway was more predominant in the individual (HG043) with high-CYP2C19 but low-3A4 levels. No particular increase in either metabolite was observed in the individual (HG023) with high levels of CYP2D6.
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Discussion |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The metabolic intrinsic clearance rates
(Vmax/Km)
indicate that liver microsomes from all three species more readily
produce a detoxication product (i.e., TCP) than an activation product (i.e., CPO). These observations are similar to previous reports on
rodents (Sultatos and Murphy, 1983; Ma and Chambers, 1995). The
clearance rates also demonstrate that pooled HLM are less active than
RLM and MLM in both desulfuration and dearylation, suggesting that less
TCP and CPO are generated in the human liver than in the rodent liver
immediately after exposure to CPS.
Consistent with data on other substrates provided by the supplier
(XenoTech, LLC) regarding gender differences in CYP activity, pooled
female HLM showed higher activities in both desulfuration and
dearylation of CPS than pooled male HLM. Note that this gender difference was demonstrated using only one pool of 10 males and 10 females, respectively. It is not known whether this difference would
also be true in a larger population. These data contrast with CYP
activities in rats because males are more active in CPS desulfuration
than females (Chambers and Chambers, 1989; Sultatos, 1991).
Our results show that human lymphoblast-expressed CYP1A2, 2B6, 2C9*1,
2C19, and 3A4 are responsible for both dearylation and desulfuration of
CPS, whereas CYP1A1, 2A6, 2C8, 2C9*2, 2D6*1, 2E1, and 4A11 did not
display detectable activities toward CPS oxidation. These results are
similar to those of a parathion metabolism study (Butler and Murray,
1997), where CYP1A2, 2B6, and 3A4 were shown to have high-desulfuration
activities toward parathion. CYP2B6 more readily generates the oxon,
similar to phenobarbital-induced CYP2B isoforms in rodents (Fabrizi et
al., 1999; Levi et al., 1988). CYP2C19 exhibits the greatest
dearylation activity and relatively low-desulfuration activity. Genetic
polymorphisms have been identified in CYP2C19 (Demorais et al., 1994),
and differential metabolic activities toward CPS by different variants
of CYP2C19 were observed in this study. Dearylation by the polymorphic
CYP2C19 alleles was significantly less than that of the wild-type
forms, which could influence the in vivo toxicity of CPS in individuals possessing these alleles.
CYP3A4 is also a highly active, although not the most active, isoform
in CPS metabolism. The fact that CYP3A4 is the most abundant CYP
isoform in human liver (Shimada et al., 1994) suggests that this
isoform plays a significant in vivo role in both desulfuration and
dearylation. CYP2C9*1 (Arg144) showed some
activities in both desulfuration and dearylation of CPS, whereas
CYP2C9*2 (Cys144), a single amino acid
difference, showed no detectable activity toward CPS. CYP2C9*1 and 1A2,
although not the most active isoforms for either desulfuration or
dearylation, may play a role in vivo. A previous study using specific
chemical inhibitors and human lymphoblastoid cell-expressed CYP2D6
suggested that CYP2D6 plays a significant role in desulfuration of
parathion, CPS, and diazinon (Sams et al., 2000). Our work with CPS did
not reveal any metabolites using human lymphoblast-expressed CYP2D6,
nor did HLM with high-CYP2D6 activity (HG023) produce a significant
amount of the oxon metabolite.
Potential differences in the human population with respect to CPS
metabolism were further examined using different individual HLM.
Individuals with varying levels of CYP2B6, 2C19, 2D6, and 3A4 were
selected to represent contrasting levels of predicted metabolic
activity. Thus, individuals (HG042 and HG112) possessing high levels of
CYP2B6 and CYP3A4 would be expected to possess greater ability to form
the desulfuration product than those (HG006, HG023, and HG043) with
lower levels of these isoforms, as observed (Table 5). Similarly,
individuals with greater levels of CYP2C19 or CYP3A4, such as HG042,
HG043, and HG112, would also be expected to produce more of the
dearylation product than those with significantly lower levels of these
isoforms (HG006 and HG023). Because individuals with contrasting levels
of CYP2B6 and 3A4 were not available, we were unable to differentiate
the extent of their contribution to desulfuration separately. However,
based on its content in human liver, CYP3A4 should contribute
significantly to both desulfuration and dearylation, as has been
previously observed (Butler and Murray, 1997; Mutch et al., 1999; Sams
et al., 2000).
Using our selection of five individuals, the variations between
individual HLM in desulfuration of CPS were around 8-fold and that for
dearylation was 3-fold and would presumably be greater if more samples
were examined. For parathion, the difference between individual HLM in
dearylation and desulfuration is as great as 10- and 16-fold,
respectively (Butler and Murray, 1997; Mutch et al., 1999).
Considerations of metabolic differences between individuals should also
consider the contributions of esterases, which are also major factors
determining the in vivo toxicities of OP compounds (Maxwell et al.,
1987; Chambers et al., 1990; Costa et al., 1990).
In conclusion, HLM use the same pathways as RLM and MLM to metabolize CPS, although activities were generally lower in humans than in rodents. From pools of 10 individuals, female HLM displayed a higher activity in CPS metabolism than male HLM. Human lymphoblast-expressed CYP1A2, 2B6, 2C9*1, 2C19, and 3A4 showed oxidation activities toward CPS, whereas no activities were detected for CYP1A1, 2A6, 2C8, 2C9*2, 2D6, 2E1, and 4A11. Although CYP2C19 and 2B6 displayed the greatest dearylation and desulfuration activities, respectively, 3A4 was highly active in both reactions. Activities of CYP2B6, 2C19, and 3A4 greatly affect CPS metabolism in HLM.
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Footnotes |
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Received March 19, 2001; accepted June 8, 2001.
This work was supported by the North Carolina Department of Agriculture (NCDA) Pesticide Environmental Trust Fund and U.S. Army Cooperative Agreement DAMD 17-00-2-0008. Preliminary studies were presented at the 10th North American International Society for the Study of Xenobiotics (ISSX) meeting in Indianapolis, 2000 and the 40th Society of Toxicology (SOT) annual meeting in San Francisco, 2001.
Dr. Ernest Hodgson, Department of Environmental and Molecular Toxicology, Box 7633, North Carolina State University, Raleigh, NC 27695. E-mail: ernest_hodgson{at}ncsu.edu
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Abbreviations |
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Abbreviations used are: CPS, chlorpyrifos; CPO, chlorpyrifos-oxon; TCP, 3,5,6-trichloro-2-pyridinol; OP, organophosphorus; HLM, human liver microsomes; RLM, rat liver microsomes; MLM, mouse liver microsomes; CYP, cytochrome P450; HPLC, high-performance liquid chromatography.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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-hydroxylase catalytic
activities, respectively, are 3.1, 36, not detectable and 2990 for
HG006 (16-year-old male), 12.2, 78.1, 160 and 4050 for HG023
(25-year-old male), 140, 3.5, 110 and 14530 for HG042 (48-year-old
female), 7.4, 212, 10.6 and 3408 for HG043 (23-year-old female), and
59.1, 260.0, 23 and 17519 for HG112 (2-year-old female) (data were
provided by GENTEST).