Battelle, Pacific Northwest Division, Chemical Dosimetry, P.O. Box 999, Richland, Washington 99352
Received November 26, 2002; accepted January 11, 2003
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ABSTRACT |
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Key Words: chlorpyrifos; diazinon; organophosphate insecticides; metabolism; liver; intestine.
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INTRODUCTION |
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A number of recent preclinical and clinical studies have demonstrated the importance of the intestines in first-pass metabolism, since both rodent and human intestinal epithelial cells (enterocytes) contain enzymes capable of metabolizing a broad range of drugs and xenobiotics (Butler and Murray, 1997; Fabrizi et al., 1999
; Hall et al., 1989
; Obach et al., 2001
; Paine et al., 1999
; Sams et al., 2000
; Watkins, 1987; and Zhang et al., 1999). In addition, P-glycoproteins (multidrug resistance proteins) on the apical borders of enterocytes are energy-dependent drug efflux pumps capable of lowering intracellular drug/chemical concentrations (Zhang and Benet, 2001
). The role of intestinal metabolism and active extrusion of absorbed drugs are of current interest since collectively they may be a major determinant of oral drug bioavailability (Zhang and Benet, 2001
). Although there has been a considerable focus on understanding the impact of intestinal metabolism on drug bioavailability, few studies have attempted to explore the influence of intestinal metabolism on the bioavailability of pesticides. In this regard, Lanning et al. (1996) investigated the interaction of CPF with P-glycoproteins and reported their upregulation in rat stomach, jejunum, and liver of rats following in vivo exposure to CPF. It was further reported that only the active metabolite of CPF, CPF-oxon, actively bound and activated P-glycoprotein, potentially contributing to the intestinal metabolism of this OP.
The metabolism of OPs to their oxon metabolites are likely mediated by CYP 3A4, 2B6 (human), 2B1/2 (rat), and 2D6 (human) (Ma and Chambers, 1995; Sams et al., 2000
; Tang et al., 2001
). Although the profile of enzymes in human intestine and liver are different, the common CYP in both the liver and small intestine is 3A4 (Obach et al., 2001
). The levels of 3A4 in the intestine may be as high as 90% of, or exceed the liver content; whereas, 2D6 and 2C are reported to be 1020% of the hepatic levels (Watkins et al., 1987
; de Waziers et al., 1990
). CYP-mediated dearylation resulting in the deactivation of CPF to TCP is mediated by CYP 2C and 3A4 (Tang et al., 2001
). B-Esterase activity is similar in both intestine and liver, whereas, the activity of PON1 is approximately 100-fold lower in intestine (Pond et al., 1995
). Thus, the metabolizing enzymes responsible for both the bioactivation and detoxification are present in the small intestine at levels lower than the liver, but still significant.
In developing physiologically based pharmacokinetic (PBPK) models, hepatic metabolism is primarily considered, and the contribution of intestinal metabolism has been ignored and "lumped" within the liver compartment. This approach has provided reasonably good estimates of OP dosimetry (Timchalk et al., 2002), since toxicity studies in animals are conducted at high doses, where gut metabolic capacity (Phases I and II) would be saturated, allowing levels of toxicant to pass to the portal circulation. However, at lower, "real world" exposure levels, intestinal metabolism could significantly impact systemic levels of these OPs, such that little if any parent compound or oxon are absorbed into the liver and distributed systemically. To assess this hypothesis, both hepatic and intestinal CYP- and PON1-mediated metabolism of CPF and DZN in microsomes prepared from liver and intestinal enterocytes of rats were evaluated. These data will subsequently be incorporated into the CPF (Timchalk et al., 2002) and DZN (Poet et al., 2002) PBPK models to assess the role of intestinal metabolism, particularly at low environmentally relevant doses.
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MATERIALS AND METHODS |
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Animals.
Adult male Sprague-Dawley rats (300400 g) were obtained from Charles River, Inc. (Raleigh, NC). Prior to use, animals were housed in solid-bottom cages with hardwood chips, and were acclimated (1 week) in a humidity- and temperature-controlled room with a 12-h light/dark cycle. Rodent feed (Purina rodent chow) and water were provided ad libitum.
Microsomes.
The rats were killed by CO2 asphyxiation, and livers and small intestine excised. Rat liver microsomes were prepared from 6 individual animals. With regard to the intestinal metabolism, the majority of CYP activity resides in the enterocytes of the small intestine (Obach et al., 2001; Wacher et al., 2001
). A section of the small intestine was excised from the base of the stomach (pyloric sphincter) to above the cecum. Enterocytes were eluted from the small intestine by the method of Fasco et al.(1993)
, and pooled from 3 animals for each microsomal sample. Microsomes were prepared from liver and enterocytes by differential ultracentrifugation by the method of van der Hoeven and Coon (1974)
. Protein concentration was determined using the BCA reagent (Pierce, Rockford, IL) with BSA as the standard, and microsomes were stored at 80°C until used.
CYP assay.
Total microsomal P450 content was determined from reduced CO difference spectra (Omura et al., 1964). Preliminary studies to confirm linearity with respect to microsomal protein concentration and time were performed (data not shown). CPF was incubated in 1 ml of 50 µM HEPES buffer containing, 15 mM MgCl2, and 1 mM EDTA. Incubations with CPF contained 1 or 3 mg of microsomal protein for hepatic and enterocyte microsomes, respectively. For DZN, enterocyte microsomal incubations contained 4 mg of protein. The samples were incubated at 37°C for 3 min and 1 mM NADPH was added to initiate the reaction, which was terminated after 10 min by the addition of 200 µl of 2.5 N acidic saturated salt solution (NaCl). Metabolism blanks with boiled microsomes were run for comparison for nonspecific breakdown of TCP, IMHP, or oxon. Preliminary studies verified that 1 mM EDTA was sufficient to block any PON1-mediated metabolism of the oxon (data not shown).
PON1 assay.
Initial studies were conducted to verify the optimal incubation conditions for protein concentration and incubation period. Microsomes were prepared in buffer lacking EDTA. Oxon (CPF-oxon or DZN-oxon) was incubated in 1 ml of buffer (0.1 M Tri-HCl buffer, pH = 8.5, with 2 mM CaCl2) containing 0.1 mg or 0.5 mg of microsomal protein for hepatic and enterocyte microsomes, respectively. The samples were incubated at 37°C for 3 min, then 10 µl of substrate in methanol was added to initiate the reaction (methanol was less than 0.1% of total volume). The reaction was terminated 2 min later by adding 200 µl 2.5 N HCl solution in concentrated salt (NaCl). Heat-inactivated microsomes were used to compare the nonspecific breakdown of the oxons.
Chloropyrifos, CPF-oxon, and TCP analysis.
Chlorpyrifos metabolism studies to date have measured the desulfuration reaction by monitoring the amount of inhibition of AChE from an exogenous source (Ma and Chambers, 1994). For this study, analytical methods were developed to directly measure the formation of oxon and TCP. Extraction of the metabolites was accomplished by adding 5 µg/ml CPF-methyl internal standard solution in toluene with the same volume of the incubation system. The solutions were mixed well on a vortex mixer, layers were separated by centrifugation, and the organic supernatant was removed. In order to place the response within the linear range of the calibration curve, the aliquot supernatant was diluted with toluene and quantitated on a Hewlet Packard 5890 gas chromatograph (GC) (Hewlett-Packard, Avondale, PA) equipped with an electron capture detector (ECD). Separation was achieved using an HP-1 capillary column (5 m x 0.53 mmi.d. x 2.65 µm film thickness: Hewlett-Packard, Avondale, PA). Helium carrier gas was used with a head pressure of 2.5 psi. The GC oven temperature program included an initial hold at 80°C for 1 min followed by a 25°C/min ramp to 220°C and then a second ramp to 270°C at 10°C/min. The final temperature was held for 1 min. The injection port was 275°C and the detector was 300°C. The retention times for TCP, CPF-oxon and CPF were approximately 3.6, 5.6, and 5.0 min, respectively.
Diazinon, DZN-oxon, and IMHP analysis.
An analytical method was developed to directly quantify the formation of DZN-oxon and IMHP. Chloropyrifos was used as an internal standard and metabolites were extracted with toluene at a 1:1 ratio (v/v). The solutions were mixed well on a vortex mixer and layers were separated by centrifugation. A 1-ml aliquot of the extract was blown down under a gentle N2 stream and reconstituted in toluene (100 µl1 ml) to place the GC response within the linear range of the calibration curve. Samples were analyzed using a Hewlett-Packard 5890 GC equipped with nitrogen phosphorus (NPD) and flame ionization (FID) detectors. Separation was achieved using a Stabilwax-DB column (15 m x 0.53 mmi.d. x 0.25 µm film thickness, Restek, Bellefonte, PA). Hydrogen was used as the carrier gas with a head pressure of 5 psi. Helium was the makeup gas for the NPD detector while N2 was the makeup gas for FID detector. A temperature program was used to ramp at a rate of 15°C/min from 65°C to 165°C, which was held for 2 min and then followed by a second ramp of 50°C/min to the final temperature of 230°C. The injection and detection temperatures were 275°C and 300°C, respectively. The retention times for DZN, DZN-oxon, and IMHP were approximately 6.0, 6.4, and 6.6 min, respectively.
Data analysis.
All hepatic microsomal incubations were conducted with n = 6 microsomal samples prepared from individual animals, and means and standard deviations were calculated. Enterocyte incubations were carried out in 2 microsomal samples, each prepared from pooled enterocytes from 3 animals. The metabolic rate constants (Vmax and Km) for metabolite formation were calculated by fitting the Michaelis-Menten equation to the data with nonlinear regression using SlideWrite Plus (Advanced Graphics Software Inc., Encinitas, CA).
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RESULTS |
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The Km for the production of the oxon is similar in enterocytes and hepatic microsomes, possibly indicating that the same enzyme or enzymes are responsible for the metabolism to CPF-oxon and DZN-oxon, respectively in these two tissues. However, the Km for the production of TCP from CPF is more than 10-fold higher in enterocyte microsomes, suggesting that the enzyme composition responsible for the metabolism of CPF to TCP may be different in enterocytes. In the case of DZN, the Km for metabolism to either DZN-oxon or IMHP is reasonably comparable in both the liver and enterocytes.
To compare the CYP metabolic capacity of enterocytes relative to hepatic microsomes, the metabolism of CPF to TCP and CPF-oxon was normalized based on P450 content (see Fig. 4). Although the metabolism of CPF and DZN are similar in liver and enterocytes, the CYP content in intestine is lower than liver (Fasco et al., 1993
; Obach et al., 2001
). In this study, total reducible CO spectra of the CYP content in enterocyte microsomes used to evaluate CPF metabolism was 10-fold lower than in microsomes prepared from liver (0.793 ± 0.140 and 0.0722 nmol/mg, respectively). However, the Vmax for enterocyte microsomal metabolism of CPF was only 45-fold lower on a per mg basis. DZN metabolism to the oxon is
10-fold lower in enterocyte microsomes than liver microsomes on a per mg basis, and DZN deactivation to IMHP is
5-fold lower. Thus, if metabolism is compared by first normalizing for CYP content, then the metabolic capacity of the enterocytes clearly exceeds that of the hepatic microsomes for CPF and is similar to or exceeds the capacity for DZN as well.
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DISCUSSION |
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A number of in vivo studies in both animals and humans suggest that the oral bioavailability of OP insecticides is fairly low. Braeckman et al.(1983) reported that only 129% of orally administered parathion was bioavailable in dogs, due to rapid first-pass metabolism. Wu et al.(1996)
conducted similar bioavailability studies with DZN and suggested that only 35% of an 80 mg/kg oral dose was systemically bioavailable. In humans, the bioavailability of orally administered CPF, based upon recovery studies and PBPK/Pharmacodynamic model simulations, ranged from as low as 2035% to 70% depending upon the physical form of the CPF when administered (Nolan et al., 1984
; Timchalk et al., 2002).
The major metabolites formed from CYP-mediated hepatic metabolism for both CPF and DZN are the inactive pyridinol/pyrimidinol with the active oxon representing the minor metabolite (see Fig. 2). These results are consistent with previous reports in both rodents and humans (Ma and Chambers, 1994
, 1995
; Fabrizi et al., 1999
; Nolan et al., 1984
Tang et al., 2001
; ). In addition, based upon the overall hepatic metabolic efficiency (pseudo-1st order rate constant: Vmax/Km), CPF will undergo more hepatic metabolism in vivo than DZN.
Microsomes derived from intestinal enterocytes demonstrated a similar CYP-mediated metabolism profile involving both desulfuration and dearylation reactions, with dearylation to the pyridinol/pyrimidinol also representing the preferred pathway for metabolism (see Fig. 3). These are the first experimental results reported to characterize the CYP-mediated metabolism of CPF and DZN in rodent enterocytes, and are consistent with the reported very high 3A subfamily activity in enterocytes (Obach et al., 2001
; Zhang and Benet, 2001
), and the relative importance of 3A for both dearylation and desulfuration metabolism of CPF and DZN (Tang et al., 2001
). In the case of enterocyte CYP metabolism, the overall metabolic efficiency for the conversion of both CPF and DZN to their corresponding oxon metabolites is
28-fold greater for CPF than DZN. This may suggest a different distribution of CYP forms within the enterocytes relative to that observed in the liver.
Although many tissues within the body are known to possess some CYP activity, the prevailing dogma is that the liver is the primary organ associated with CYP metabolism and is responsible for first-pass metabolism of chemicals and drugs absorbed from the gastrointestinal tract. It is clear from recent studies that enterocytes within the small intestine also possess CYP metabolic capacity, and in the case of some enzymes (i.e., 3A4) the concentration within the enterocytes is comparable to the liver (Obach et al., 2001; Wacher et al., 2001
; Zhang and Benet, 2001
). In comparing the CYP metabolism of CPF with DZN in the current study on a mg-protein basis, the hepatic microsomes are slightly more efficient at metabolizing CPF to CPF-oxon (2-fold) and clearly better at metabolizing to TCP (61-fold); however, if the metabolism is compared on a nmol P450 basis it is clear that the overall CYP metabolic rate for the enterocytes exceeds that of the liver microsomes (see Fig. 4
). P-Glycoprotein efflux of the oxon metabolite may also contribute to lowering the bioavailability of these OP insecticides. Although the parent CPF apparently does not interact with P-glycoproteins, the oxon does (Lanning et al., 1996; Yang et al., 2001
). Thus, a fraction of the oxon produced in the enterocytes may be transported back into the lumen of the small intestine, thereby, never reaching the systemic circulation. Future studies are needed to evaluate this hypothesis and determine the potential role of P-glycoprotein efflux (cell to lumen) of oxon within intestinal enterocytes.
Since PON1 is an important detoxification pathway for the oxon (Main, 1956; Costa et al., 1990
; Li et al., 1993
; 1995
), the PON1-mediated metabolism of CPF-oxon and DZN-oxon were evaluated in microsomes derived from intestinal enterocytes, the results clearly demonstrate that the enterocytes possess PON1 activity and, based on the comparable Km values between the liver and enterocytes, they suggest that these are similar enzymes. Although the Km values were comparable between the liver and enterocyte microsomes, it was clear that the Vmax for the liver exceeded that of enterocytes, resulting in a much higher metabolic efficiency within the liver for CPF-oxon (255-fold), and DZN-oxon (69-fold) metabolism. Pond et al.(1995)
compared the PON1 activity in numerous tissues including the small intestines of rats, using CPF-oxon as substrate. They reported
113-fold greater activity in the liver versus the small intestines, which is consistent with the lower activity found in the current study. Although the intestines contain significantly less PON1 activity than the liver, the results still suggest that the oxon metabolites can be subjected to significant detoxification within the enterocytes. In addition, the intestines are reported to have high CaE activity (Pond et al., 1995
) that can stochiometrically bind to and detoxify oxon. The importance of CaE detoxification of OP insecticides has been previously established (Chanda et al., 1997; Clement, 1984
; Fonnum et al., 1985
; Maxwell, 1992
) and may be of particular importance when considering low-level environmental exposures.
Although the results of the current in vitro study clearly suggests that intestinal metabolism of CPF and DZN could modify in vivo bioavailability, there is still a question as to how important intestinal metabolism is relative to the contribution of the liver. In this regard, it has been demonstrated using in vivo animal studies and human clinical evaluations that the inhibition or induction of intestinal CYP metabolism can clearly modify the oral bioavailability of a number of drugs including, cyclosporine, tacrolimus, and digoxin (Floren et al., 1997; Gomez et al., 1995
; Paine et al., 1999
; Salphati and Benet, 1998
; Zhang and Benet, 2001
). Hence, it is not unreasonable to anticipate that intestinal CYP metabolism would likewise impact OP bioavailability as a result of CYP-mediated metabolism (i.e., activation and detoxification). Second, although there was approximately a 10-fold lower total P450 per mg of protein in the intestinal microsomes prepared in this study than in liver microsomes, in rats the liver is
4% of the total body volume whereas the small intestines comprise
2.5% (Schoeffner et al., 1999
); hence the overall capacity of the small intestine for metabolism may be substantial. Third, the intestines, and specifically the enterocytes lining the gut walls, are designed to maximize surface area (numerous microvilli) to facilitate absorptive processes including the uptake of OP insecticides. Once the OPs are within the enterocytes, based upon the in vitro metabolism results, it is reasonable to anticipate that they will be subjected to both CYP- and PON1-mediated metabolism prior to entering the liver via the portal circulation. Although the current in vitro studies support a working hypothesis that intestinal metabolism can contribute to the oral bioavailability of OP insecticides, additional in vivo kinetic studies are needed to substantiate the role of enterocyte metabolism in modifying the oral bioavailabilty of CPF and DZN, particularly at relevant low environmental exposure levels. In addition, the correlation between rat and human metabolism and intestinal absorption needs to be assessed.
In summary, the inhibition of AChE by CPF and DZN results in the accumulation of acetylcholine at the neuronal junctions, which is the primary toxic mode of action for these OPs. Although the parent OPs have some inhibitory potential, the primary toxicants are the oxon metabolites. Chlorpyrifos and DZN are bioactivated by CYP enzymes to the oxon metabolites and deactivated by both CYPs and PON1 to pyridinol and pyrimidinol, respectively (TCP from CPF and IMHP from DZN). The balance between bioactivation and detoxification will drive the toxicity from OP exposures. At low oral dose exposures, metabolism by CYPs and PON1 in intestine and liver may remove the active moiety from the circulation prior to systemic exposure. These results suggest that, in the rat, intestinal metabolism should be considered when evaluating biological response, particularly to low doses. The impact of intestinal metabolism may be further evaluated through appropriate in vivo studies and by incorporating an intestinal metabolic compartment into current PBPK models for both CPF and DZN (Poet et al., 2002; Timchalk et al., 2002).
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at Battelle, Pacific Northwest Division, Chemical Dosimetry, MSIN P7-59, P.O. Box 999, Richland, Washington 99352. Fax: (509) 376 9064. E-mail: torka.poet{at}pnl.gov.
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