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Estimating constants for metabolism of atrazine in freshly isolated rat hepatocytes by kinetic modeling
Abstract
This study estimated the kinetic constants for oxidative metabolism of atrazine (ATRA) and its chlorotriazine (Cl-TRI) metabolites, 2-chloro-4-ethylamino-6-amino-1,3,5-triazine (ETHYL), 2-chloro-4-amino-6-isopropylamino-1,3,5-triazine (ISO), and diaminochlorotriazine (DACT), using freshly isolated rat hepatocytes. Hepatocytes were incubated with 1.74, 44, 98, and 266 microM ATRA. Disappearance of ATRA and formation of the Cl-TRI metabolites were quantified over 90 min. At all incubation concentrations, ATRA was preferentially metabolized to ETHYL, producing ETHYL concentrations approximately 6 times higher than those of ISO. DACT concentrations peaked at 44 microM ATRA and decreased with increasing incubation concentrations, indicating non-linear metabolic behavior of ATRA with respect to DACT formation. A series of kinetic models were developed from these data to describe the dose and time-dependent oxidative metabolism of ATRA and the Cl-TRI metabolites. An integrated model for all the chloro-triazines included multi-substrate competitive inhibition of metabolism to describe the non-linear behavior of DACT production in relation to ATRA while simultaneously simulating the time-course behavior of the Cl-TRIs at all four ATRA concentrations. The maximal metabolic rate (V(max)) of ATRA metabolism and the Michaelis-Menten constant (K(M)) for the reaction were 1.6 microM/min and 30 microM, respectively. V(max) and K(M) values for ETHYL and ISO metabolism to DACT were also estimated using this modeling approach.
... The previously-published PBPK model (McMullin et al., 2007a) was re-parameterized using new in vitro and in vivo data to produce a model that 1) provided more accurate estimates of the absorbed dose, and 2) reliably predicted measured plasma concentrations of the (Foradori et al., 2014). Even though the urinary DEA, DIA and DACT clearance rates in this model were informed by human TCT elimination data, the model could be further improved if clearance rates for conjugated metabolites of ATZ, DEA, DIA and DACT were available. ...
... The in vitro oxidative metabolism rates for ATZ to DEA, DIA and DACT were determined in rat and human hepatocytes(McMullin et al., 2007a).McMullin et al. incubated intact rat hepatocytes with ATZ for 90 minutes at initial concentrations of 1.74, 44, 98 or 266 µM and measured changes in the concentration of the chlorometabolites over time. In a new study, rat or human hepatocytes (0.5 x 10 6 hepatocytes per mL media) were incubated with ATZ at nominal concentrations of 0.5, 1.0 or 1.7 µM, and average initial concentrations of 0.45, 1.26 by guest on January 25240 minutes after exposure initiation. ...
... To account for the reduced conversion rates to DACT observed at higher incubation concentrations, competitive inhibition information between substrates for ATZ, DIA and DEA was included in the model. Michaelis-Menten affinity constants (Km) for ATZ, DEA and DIA were 30, 13 and 13µm, respectively(McMullin et al., 2007a). Maximum metabolism rates (Vmax) were estimated serially; then the fraction of either DIA or DEA produced from ATZ was estimated. ...
The previously-published PBPK model for atrazine (ATZ), deisopropylatrazine (DIA), deethylatrazine (DEA) and diaminochlorotriazine
(DACT), which collectively comprise the total chlorotriazines (TCT) as represented in this study, was modified to allow for
scaling to humans. Changes included replacing the fixed dose-dependent oral uptake rates with a method that represented delayed
absorption observed in rats administered ATZ as a bolus dose suspended in a methylcellulose vehicle. Rate constants for metabolism
of ATZ to DIA and DEA, followed by metabolism of DIA and DEA to DACT, were predicted using a compartmental model describing
the metabolism of the chlorotriazines by rat and human hepatocytes in vitro. Overall, the model successfully predicted both the four-day plasma time-course data in rats administered ATZ by bolus dose
(3, 10 and 50 mg/kg/day) or in the diet (30, 100 or 500 ppm). Simulated continuous daily exposure of a 55-kg adult female
to ATZ at a dose of 1.0 μg/kg/day resulted in steady-state urinary concentrations of 0.6, 1.4, 2.5 and 6.0 μg/L for DEA, DIA,
DACT and TCT, respectively. The TCT (ATZ + DEA + DIA + DACT) human urinary biomonitoring equivalent (BE) concentration following
continuous exposure to ATZ at the chronic point of departure (POD = 1.8 mg/kg/day), was 360.6 μg/L.
... A starting point for the estimation of the metabolic rate constants of ATR to DE, and ATR to DIP was an in vitro study with mouse liver microsomes (Hanioka et al., 1999b) as in vitro techniques have been shown to be a valuable a priori parameterization, with in vitro metabolic rates typically being 2-to 3-fold lower than corresponding in vivo values (Kramer et al., 2001). There are no in vitro data on the metabolic rate constants of DE to DACT and DIP to DACT, as, in vitro, the DE/DIP to DACT conversion is minimal across mammals (Lang et al., 1996;McMullin et al., 2007a;Joo et al., 2010), including conversion by mouse microsomal preparations (Hanioka et al., 1998(Hanioka et al., , 1999aRoss and Filipov, 2006). In the present mouse model, the in vivo hepatic oxidative metabolic parameters of ATR to DE and ATR to DIP were estimated by manually adjusting based on the corresponding in vitro metabolic rates, and optimal simulations of plasma concentrations for ATR and its Cl-TRIs were obtained when the in vivo metabolic rates were 2-fold higher than the in vitro metabolic rates. ...
... The PBPK model predictions of plasma, liver, and brain dosimetry of ATR and its Cl-TRIs were compared to measured data of mice dosed orally with 250 mg/kg ATR, as shown in Fig. 2. Initially, we set all the absorption rate constants (K1, K2, and K3) to be 1 and incorporated the published in vitro metabolic rate constants of ATR to DE, and ATR to DIP determined using liver microsomes (Hanioka et al., 1999b) into the mouse model. The result was an overestimation of plasma ATR levels and an underestimation of plasma Cl-TRIs levels, which was the same problem that McMullin et al. (2007b) met after incorporation of the in vitro metabolic rate of ATR determined using rat hepatocytes (McMullin et al., 2007a) into the rat model. Next, we increased the in vivo metabolic rates of ATR to DE, and ATR to DIP 2-fold; incorporated the visually estimated metabolic rate constants of DE and DIP to DACT for the mouse; and slightly adjusted the absorption rate constants based on relevant data in the rat model (McMullin et al., 2003). ...
... However, they can be engineered and reprogrammed to do so, which is another strategy for ATR elimination from the environment that may be employed in the future (Sinha et al., 2010). Based on these observations and the fact that ATR is metabolized similarly by rat, mouse, pig, and human liver microsomes, i.e., DE and DIP are the two major in vitro cytochrome P450 metabolites (Lang et al., 1996;Hanioka et al., 1998Hanioka et al., , 1999aRoss and Filipov, 2006;McMullin et al., 2007a;Joo et al., 2010), we did not include intestinal metabolism in our model. Rather, we incorporated in vivo metabolic rates of ATR that are 2-fold higher than the reported mouse-specific in vitro values. ...
Atrazine (ATR) is a chlorotriazine herbicide that is widely used and relatively persistent in the environment. In laboratory rodents, excessive exposure to ATR is detrimental to the reproductive, immune, and nervous systems. To better understand the toxicokinetics of ATR and to fill the need for a mouse model, a physiologically based pharmacokinetic (PBPK) model for ATR and its main chlorotriazine metabolites (Cl-TRIs) desethyl atrazine (DE), desisopropyl atrazine (DIP), and didealkyl atrazine (DACT) was developed for the adult male C57BL/6 mouse. Taking advantage of all relevant and recently made available mouse-specific data, a flow-limited PBPK model was constructed. The ATR and DACT sub-models included blood, brain, liver, kidney, richly and slowly perfused tissue compartments, as well as plasma protein binding and red blood cell binding, whereas the DE and DIP sub-models were constructed as simple five-compartment models. The model adequately simulated plasma levels of ATR and Cl-TRIs and urinary dosimetry of Cl-TRIs at four single oral dose levels (250, 125, 25, and 5mg/kg). Additionally, the model adequately described the dose dependency of brain and liver ATR and DACT concentrations. Cumulative urinary DACT amounts were accurately predicted across a wide dose range, suggesting the model's potential use for extrapolation to human exposures by performing reverse dosimetry. The model was validated using previously reported data for plasma ATR and DACT in mice and rats. Overall, besides being the first mouse PBPK model for ATR and its Cl-TRIs, this model, by analogy, provides insights into tissue dosimetry for rats. The model could be used in tissue dosimetry prediction and as an aid in the exposure assessment to this widely used herbicide.
... To address this deficiency, in vitro studies in freshly isolated rat hepatocytes were conducted that examined the dose-dependent metabolic behavior of ATRA and the Cl-TRI metabolites. From these data, Michaelis-Menten (M-M) parameters for the oxidative metabolism of ATRA to the chlorinated metabolites were estimated using a kinetic modeling approach (McMullin et al., 2007). The objective of the modeling approach pre-sented in this paper was to integrate these in vitro kinetic parameters with in vivo data on the time-course disposition of the individual Cl-TRI metabolites in the plasma to evaluate the role of absorption and oxidative metabolism in determining the kinetics of individual Cl-TRI metabolites in the rat. ...
... Hepatic metabolism of ATRA to the mono-dealkylated metabolites was described by M-M kinetics similar to the metabolism of the mono-dealkylated metabolites in the models for ETHYL and ISO. Results from recent in vitro studies evaluating the oxidative metabolism of ATRA suggested that competitive metabolic inhibition of the mono-dealkyalted metabolites occurred at high incubation concentrations of ATRA (McMullin et al., 2007). This possibility was examined in vivo by including metabolic inhibition of ISO and ETHYL metabolism to DACT into the description of saturable, oxidative metabolism. ...
... The chemical specific parameters estimated in the DACT, ETHYL and ISO models, the volume of distribution determined in the DACT model and the hepatic oxidative metabolic rate constants for each metabolite were carried over to the ATRA model. In vitro estimated parameters for hepatic oxidative metabolism of ATRA to the mono-dealkylated metabolites (V max atra, K M atra) and the fraction of ATRA metabolized to ISO (frac) (McMullin et al., 2007) were also incorporated into the ATRA model. This approach minimized the number of parameters that needed to be estimated simultaneously in each model and provided a common set of biologically realistic compound specific values that described the individual Cl-TRI plasma profiles following each dosing situation (Table 1). ...
Atrazine (ATRA) is metabolized by cytochrome P450s to the chlorinated metabolites, 2-chloro-4-ethylamino-6-amino-1,3,5-triazine (ETHYL), 2-chloro-4-amino-6-isopropylamino-1, 3, 5-triazine (ISO), and diaminochlorotriazine (DACT). Here, we develop a set of physiologically based pharmacokinetic (PBPK) models that describe the influence of oral absorption and oxidative metabolism on the blood time course curves of individual chlorotriazines (Cl-TRIs) in rat after oral dosing of ATRA. These models first incorporated in vitro metabolic parameters to describe time course plasma concentrations of DACT, ETHYL, and ISO after dosing with each compound. Parameters from each individual model were linked together into a final composite model in order to describe the time course of all 4 Cl-TRIs after ATRA dosing. Oral administration of ISO, ETHYL and ATRA produced double peaks of the compounds in plasma time courses that were described by multiple absorption phases from gut. An adequate description of the uptake and bioavailability of absorbed ATRA also required inclusion of additional oxidative metabolic clearance of ATRA to the mono-dealkylated metabolites occurring in GI a tract compartment. These complex processes regulating tissue dosimetry of atrazine and its chlorinated metabolites likely reflect limited compound solubility in the gut from dosing with an emulsion, and sequential absorption and metabolism along the GI tract at these high oral doses.
... The major CYP-derived metabolites of atrazine are deethylatrazine (DEA) and deisopropylatrazine (DIA). Both are metabolized to diamino-s-chlorotriazine (DACT) [8,19], which is the major atrazine metabolite detected in vivo in rats [20]; however, the enzyme effecting its formation remains unknown [16,21]. Between 8-12 metabolites of atrazine conjugated [22] via glutathione s-transferases [23] have been identified in urinary metabolites profile [24]. ...
Background
The increasing prevalence of male infertility and the declining trend in sperm quality has been associated to compounds known as “endocrine-disruptors” . The proven endocrine-disrupting effects of atrazine and propazine herbicides led us to conduct long-term research based on highly accurate specific analytical methods with a view to confirming the suspected association. Among the proposed developments was a sensitive analytical method for the simultaneous determination of three metabolites of atrazine and propazine.
Results
In this work, the method was for first time used for the chromatographic separation and determination of deethyl- and deisopropyl-atrazine (DEA and DIA, respectively) and propazine-2-hydroxy (PP-2OH) in human seminal plasma by LC–ESI-MS/MS using deuterated atrazine (d5-AT) as internal standard (IS). Chromatographic and mass spectrometric conditions such as the mobile phase composition and flow-rate, injected volume, dry gas source temperature and flow-rate, nebulizer pressure and capillary voltage were all carefully optimized. Analytes were identified and quantified by using the multiple reaction monitoring (MRM) mode as applied to positive ions ([M + H] ⁺ ). Transitions at three different m / z values for each analyte were selected from precursor ions, and the 212.1 → [128] ⁺ , 188.1 → [146] ⁺ and 174.1 → [68.1] ⁺ transitions for PP-2OH, DEA and DIA, respectively, were found to be quantitative. The proposed method was validated in terms of precision (repeatability and reproducibility), linear range (10–240 ng mL –1 ), limit of detection (150–210 pg mL –1 ), and quantification (500–700 pg mL –1 ), recovery, accuracy and matrix effects on extracts from variably treated seminal plasma samples. The overall analytical method was successfully applied to human seminal plasma samples from volunteers. PP-2OH was found at concentrations from 1.10 to 11.3 ng mL –1 in four of the six samples, and so was DIA at 9.60 ng mL –1 in one.
Conclusions
These results are suggestive of bioaccumulation of the target analytes in humans. Untargeted analytes including suspected parent molecules (atrazine and propazine) and other ions [viz., deethyldeisopropyl-atrazine (DD) and diamino-s-chlorotriazine (DACT)] were also detected under the working conditions used. These results may open up new prospects for as yet very incipient research into the bioaccumulation of endocrine disruptors in seminal plasma.
Graphical Abstract
... Un autre type de couplage peut être réalisé. Le modèle PBPK du composé parent peut être relié au niveau des sites de métabolisme à un modèle décrivant la cinétique du métabolite (Brochot et al. 2007;Gill et al. 2013;Heredia-Ortiz et al. 2011;McMullin et al. 2007a). Le modèle du métabolite peut être de type physiologique et décrire l'ensemble des processus ADME. ...
Population is largely exposed to pyrethroids, an insecticide family. The parent compound is suspected to induce neuronal and hormonal modifications in humans. Among this family, permethrin, a mixture of isomers cis/trans, is mainly used in house tratments. In this PhD project, we developed a PBK model of permethrin and some urinary metabolites uses as biomarkers of exposure. The matabolic interactions between the two isomers were also evaluated. A three steps strategy was followed. An analytical method by GC-MS/MS was developed to measure these compounds simultaneously in the different matrices. A PBPK of permethrin in rat was associated to a reduced PBPK model of DCCA and a 2-compartment model of 4'-OH-PBA and 3-PBA. The toxicokinetics parameters of each compound were estimated in a Bayesian framework from in vivo experiments in rats orally dosed with 25 mg/kg of cis- or trans permethrin. The PBPK model of permethrin was validated on the kinetic data of a mixture of permethrin. The hepatic metabolism was quantified in humans in primary hepatocytes in optimal conditions for in vitro-in vivo extrapolation, by incubating the isomers separately and as a mixture. This work underlines that a general PBPK model for Type 2 pyrethroids can be considered for the parent compound The lack of interaction between isomers during in vitro experiments and the validation of the PBPK model of permethrin could simplify the characterization of the exposure to a mixture of pyrethroids.
... quantities during mouse hepatic microsomal incubations (Ross and Filipov, 2006) or by freshly isolated rat hepatocytes (McMullin et al., 2007). The higher levels of DE compared with DIP in mouse urine that we noted are consistent with the metabolite profile observed in human urine of ATR-exposed subjects (Barr et al., 2007 ). ...
2-Chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine (atrazine, ATR) is a toxicologically important and widely used herbicide. Recent studies have shown that it can elicit neurological, immunological, developmental, and biochemical alterations in several model organisms, including in mice. Because disposition data in mice are lacking, we evaluated ATR's metabolism and tissue dosimetry after single oral exposures (5-250 mg/kg) in C57BL/6 mice using liquid chromatography/mass spectrometry (Ross and Filipov, 2006). ATR was metabolized and cleared rapidly; didealkyl ATR (DACT) was the major metabolite detected in urine, plasma, and tissues. Plasma ATR peaked at 1 h postdosing and rapidly declined, whereas DACT peaked at 2 h and slowly declined. Most ATR and metabolite residues were excreted within the first 24 h. However, substantial amounts of DACT were still present in 25- to 48-h and 49- to 72-h urine. ATR reached maximal brain levels (0.06-1.5 microM) at 4 h (5-125 mg/kg) and 1 h (250 mg/kg) after dosing, but levels quickly declined to <0.1 microM by 12 h in all the groups. In contrast, strikingly high concentrations of DACT (1.5-50 microM), which are comparable with liver DACT levels, were detectable in brain at 2 h. Brain DACT levels slowly declined, paralleling the kinetics of plasma DACT. Our findings suggest that in mice ATR is widely distributed and extensively metabolized and that DACT is a major metabolite detected in the brain at high levels and is ultimately excreted in urine. Our study provides a starting point for the establishment of models that link target tissue dose to biological effects caused by ATR and its in vivo metabolites.
Environmental contamination by chlorotriazines has been evidenced in mother-child cohort suggesting more detailed risk assessment of these compounds in drinking water. Exposure of rodents to atrazine has been associated to alterations of endocrine and reproductive functions by disrupting neuroendocrine control at hypothalamus level. Perinatal exposure to low doses of atrazine has been associated to reproductive dysfunction, and to behavioral abnormalities in adult exposed during embryogenesis. The objectives of the current investigation were to (1) evaluate the influence of physico-chemical properties of chlorotriazines on tissue distribution in pregnant rats and in fetuses, (2) gain a better understanding of fetal distribution of chlorotriazines in specific tissues, particularly in brain.
Serial blood samples were obtained from pregnant rats after administration of atrazine (ATZ), propazine (PRO) and simazine (SIM) via oral route at a dose of 10 mg/kg from day 15 to day 19. Maternal and fetal tissues were harvested at day 20, 24 hrs after the last dosing.
The metabolic extraction ratio was estimated to 87% suggesting a significant first-pass effect explaining the low oral bioavailability. Blood exposure to parent compounds (ATZ, PRO and SIM) was negligible (lower than 5%) compared to metabolite exposure. The main metabolite exposure involved diamino-s-chlorotriazine (DACT), ranging from 60 to 90% depending on the molecules administered. A correlation between tissue-to-blood ratio and physico-chemical descriptors were observed for fat and mammary gland tissues but not for brain in adult rats. A more pronounced distribution in fetal brain was observed for ATZ and PRO, the two most lipophilic compounds.
A ten-channel single-mode wavelength division demultiplexer operating at 740, 750, 760, 770, 780, 790, 800, 810, 820 and 830 nm with diffraction angles varying from 16 degree(s) to 52 degree(s) and using a graded index (GRIN) polymer waveguide is reported for the first time. Diffraction efficiency up to 60% was measured. The wavelength spreading of the Ti:Al2O3 laser (approximately equals 4 nm, 3 dB bandwidth) causes an average crosstalk figure of -21 dB. The beamwidth of the diffracted signal as a function of the input beam width, the grating interaction length, and the diffraction angle are considered. Occurrence of maximum values are discussed. A waveguide lens is needed to efficiently couple the diffracted light into an output fiber whenever the diffracted beam size is beyond the core diameter of the fiber involved.
A new technique employing continuous recirculating perfusion of the rat liver in situ, shaking of the liver in buffer in vitro, and filtration of the tissue through nylon mesh, results in the conversion of about 50% of the liver into intact, isolated parenchymal cells. The perfusion media consist of: (a) calcium-free Hanks' solution containing 0.05% collagenase and 0.10% hyaluronidase, and (b) magnesium and calcium-free Hanks' solution containing 2 mM ethylenediaminetetraacetate. Biochemical and morphologic studies indicate that the isolated cells are viable. They respire in a medium containing calcium ions, synthesize glucose from lactate, are impermeable to inulin, do not stain with trypan blue, and retain their structural integrity. Electron microscopy of biopsies taken during and after perfusion reveals that desmosomes are quickly cleaved. Hemidesmosome-containing areas of the cell membrane invaginate and appear to pinch off and migrate centrally. Tight and gap junctions, however, persist on the intact, isolated cells, retaining small segments of cytoplasm from formerly apposing parenchymal cells. Cells which do not retain tight and gap junctions display swelling of Golgi vacuoles and vacuoles in the peripheral cytoplasm. Cytoplasmic vacuolization in a small percentage of cells and potassium loss are the only indications of cell injury detected. By other parameters measured, the isolated cells are comparable to normal hepatic parenchymal cells in situ in appearance and function.
Benzene, an important industrial solvent, is also present in unleaded gasoline and cigarette smoke. The hematotoxic effects of benzene in humans are well documented and include aplastic anemia, pancytopenia, and acute myelogenous leukemia. However, the risks of leukemia at low exposure concentrations have not been established. A combination of metabolites (hydroquinone and phenol, for example) may be necessary to duplicate the hematotoxic effect of benzene, perhaps due in part to the synergistic effect of phenol on myeloperoxidase-mediated oxidation of hydroquinone to the reactive metabolite benzoquinone. Because benzene and its hydroxylated metabolites (phenol, hydroquinone, and catechol) are substrates for the same cytochrome P450 enzymes, competitive interactions among the metabolites are possible. In vivo data on metabolite formation by mice exposed to various benzene concentrations are consistent with competitive inhibition of phenol oxidation by benzene. In vitro studies of the metabolic oxidation of benzene, phenol, and hydroquinone are consistent with the mechanism of competitive interaction among the metabolites. The dosimetry of benzene and its metabolites in the target tissue, bone marrow, depends on the balance of activation processes such as enzymatic oxidation and deactivation processes such as conjugation and excretion. Phenol, the primary benzene metabolite, can undergo both oxidation and conjugation. Thus the potential exists for competition among various enzymes for phenol. Zonal localization of phase I and phase II enzymes in various regions of the liver acinus also impacts this competition. Biologically based dosimetry models that incorporate the important determinants of benzene flux, including interactions with other chemicals, will enable prediction of target tissue doses of benzene and metabolites at low exposure concentrations relevant for humans.
The chloro-S-triazine herbicides (i.e., atrazine, simazine, cyanazine) constitute the largest group of herbicides sold in the United States. Despite their extensive usage, relatively little is known about the possible human-health effects and mechanism(s) of action of these compounds. Previous studies in our laboratory have shown that the chlorotriazines disrupt the hormonal control of ovarian cycles. Results from these studies led us to hypothesize that these herbicides disrupt endocrine function primarily through their action on the central nervous system. To evaluate this hypothesis, we examined the estrogen-induced surges of luteinizing hormone (LH) and prolactin in ovariectomized Sprague-Dawley (SD) and Long-Evans hooded (LE) rats treated with atrazine (50-300 mg/kg/day, by gavage) for 1, 3, or 21 days. One dose of atrazine (300 mg/kg) suppressed the LH and prolactin surge in ovariectomized LE, but not SD female rats. Atrazine (300 mg/kg) administered to intact LE females on the day of vaginal proestrus was without effect on ovulation but did induce a pseudopregnancy in 7 of 9 females. Three daily doses of atrazine suppressed the estrogen-induced LH and prolactin surges in ovariectomized LE females in a dose-dependent manner, but this same treatment was without effect on serum LH and prolactin in SD females. The estrogen-induced surges of both pituitary hormones were suppressed by atrazine (75-300 mg/kg/day) in a dose-dependent manner in females of both strains evaluated after 21 days of treatment. Three experiments were then performed to determine whether the brain, pituitary, or both organs were the target sites for the chlorotriazines. These included examination of the ability of (1) the pituitary lactotrophs to secrete prolactin, using hypophyosectomized females bearing pituitary autotransplants (ectopic pituitaries); (2) the synthetic gonadotropin-releasing hormone (GnRH) to induce LH secretion in females treated with high concentrations of atrazine for 3 days; and (3) atrazine (administered in vivo or in vitro) to suppress LH and prolactin secretion from pituitaries, using a flow-through perifusion procedure. In conclusion, the results of these studies demonstrate that atrazine alters LH and prolactin serum levels in the LE and SD female rats by altering the hypothalamic control of these hormones. In this regard, the LE female appeared to be more sensitive to the hormone suppressive effects of atrazine, as indicated by the decreases observed on treatment-day 3. These experiments support the hypothesis that the effect of atrazine on LH and prolactin secretion is mediated via a hypothalamic site of action.
The objective of this study was to develop an analytical method to detect and quantitate the chlorotriazine herbicide atrazine (ATRA), and its chlorinated metabolites [desethylatrazine (DE-ATRA), desisopropylatrazine (DI-ATRA), and diaminochlorotriazine (DACT)] in plasma. Control plasma separated from whole rat blood was fortified with known concentrations of ATRA, DE-ATRA, DI-ATRA, and DACT. These compounds were extracted from the plasma using a liquid-liquid extraction technique, and the resulting extracts were derivatized with tetrabutyl ammonium hydroxide and methyl iodide to produce methylated derivatives of ATRA and its chlorinated metabolites. Derivatized samples and standards were analyzed using gas chromatography-mass spectrometry with selected ion monitoring. Recoveries of fortified plasma samples ranged from 84% to 97% and were validated to 100 ng/mL. This analytical method was subsequently verified in a small-scale animal study to determine time course concentrations of chlorotriazines in plasma following a single oral gavage dose of ATRA to female Sprague Dawley rats.
Celecoxib, a selective cycloxigenase-2 (Cox-2) inhibitor, is useful to treat osteoarthrosis and rheumatoid arthritis. After an oral administration, it is metabolised by the liver through the cytocrome P450 2C9 pathway. Inhibitors and inductors of this enzymatic pathways, such as fluconazole, cardiovascular drugs, psychoactive drugs, opioids and some neuroleptics and antidepressants, may interfere with celecoxib biodisponibility so that its dose should be adjusted.
The metabolism of atrazine and 6 possible metabolites by rat liver subcellular fractions was studied in vitro. The dealkylation reaction predominated over the conjugation reaction with glutathione; the isopropyl group being more easily dealkylated than the ethyl group. With the compounds investigated, the reactions involved dealkylation in the microsomal fraction and conjugation with glutathione in the soluble fraction. All of the chloro-s-triazines were able to form conjugates with glutathione. No evidence for the dechlorination of the chloro-s-triazines to hydroxy-s-triazines was observed in vitro.
The metabolism of drugs in isolated rat hepatocytes has been investigated. Drugs which are metabolized by aromatic hydrolation, aliphatic hydroylation, N-demethylation, or glucuronidation have been used as substrates. With some substrates the rate of metabolism in isolated hepatocytes compares with that in hepatic 900g supernatant fraction or microsomes, but other substrates are metabolized at a slower rate in isolated hepatocytes. For example, the rate of butamoxane hydroxylation in isolated hepatocytes is slower than that in microsomes. However, the rate of hydroxylation is hepatocytes is identical to that in perfused liver. The metabolism of drugs in isolated hepatocytes correlates with in vivo drug metabolism better than does metabolism in the hepatic 9000g supernatant fraction or microsomes.
Publisher Summary This chapter discusses preparation of isolated rat liver cells. The early mechanical and chemical methods for liver-cell preparation were relatively successful in converting liver tissue to a suspension of isolated cells. The successful preparation of intact liver cells by perfusion with collagenase is technically quite difficult. The major method for preparation of nonparenchymal liver cells is based on the selective sensitivity of parenchymal cells toward proteases. By incubation of rat liver minces with pronase, most of the liver parenchyma is digested, while nonparenchymal cells remain intact and can be recovered from the incubate. Similar results have been reported with trypsin digestion of collagenase-dispersed liver minces, but pronase appears to be more effective. The most common procedure is to perfuse the liver briefly with pronase before it is minced and incubated with the enzyme. Such direct pronase methods have been used by several investigators with yields of nonparenchymal liver cells reported to be in the range 2–15 × 10 6 cells/gm liver.
In vitro metabolism of trichloroethylene (TRI) and trichloroethanol (TCE) was investigated using liver microsomes from control and ethanol-, phenobarbital (PB)-, and 3-methylcholanthrene (MC)-treated rats. At least three forms of enzymes were involved in TRI metabolism. One was a low-Km type normally existing in microsomes from control rats. The ethanol-inducible enzyme was found to be catalytically identical to this low-Km isozyme. Another was a high-Km type which was induced exclusively by PB, and a third was an MC-inducible isozyme with a Km value between those of ethanol- and PB-inducible isozymes. Although MC treatment did not affect the rate of TRI metabolism in vitro, both ethanol and PB treatment markedly enhanced the metabolism. Ethanol-induced enhancement was different from PB-induced enhancement in that ethanol enhanced the metabolism predominantly at low substrate concentrations, whereas PB did so at high concentrations. In addition, TRI metabolism with enzymes from ethanol-treated rats was inhibited by the substrate itself at high concentrations. MC treatment of rats had little or no influence on the rate of TCE metabolism in vitro, whereas both ethanol and PB enhanced the microsomal conversion of TCE to chloral hydrate. As in the case of TRI metabolism, ethanol induced a microsomal TCE-metabolizing enzyme of low Km, whereas PB preferentially induced an enzyme of high Km.
n-Hexane-induced neuropathy is thought to be mediated through metabolism to 2,5-hexanedione (2,5HD), but there is no information on target tissue concentrations of 2,5HD after inhalation exposure to a range of n-hexane vapor concentrations. Male Fischer-344 rats were exposed to 500, 1000, 3000, and 10,000 ppm n-hexane in the air. n-Hexane and its metabolites methyl-n-butyl ketone (MBK), 2,5-dimethylfuran (DMFU), 2,5HD, 2-hexanol, and 1-hexanol were quantified by in several tissues at time intervals during and following a single 6-hr exposure to n-hexane. Urinary metabolites were quantified following a single n-hexane exposure. n-Hexane concentrations achieved an apparent steady state within 2 hr in all tissues. Peak blood concentrations of n-hexane were 1, 2, 8, and 21 μg/ml and peak sciatic nerve concentrations were 12, 48, 130, and 430 μg/g at 500, 1000, 3000, and 10,000 ppm, respectively. The half-lives of n-hexane and MBK were on the order of 1 to 2 hr in all tissues except the kidneys (). The data showed a complex relationship between n-hexane exposure and peak concentrations of the remaining metabolites. Tissue concentrations of 2,5HD, the metabolite of greatest toxicological interest, were not proportional to dose. Highest 2,5HD concentrations were found following exposure to 1000 ppm n-hexane in the blood, kidneys, and sciatic nerve (6.1, 55, and 25 μg/g, respectively). The data indicated that the metabolism and elimination of n-hexane were dependent upon exposure concentration. Consequently, n-hexane exposure concentration cannot be directly correlated with tissue 2,5HD concentrations, and severity of neuropathy may not be directly related to n-hexane exposure concentration.
Furan is both hepatotoxic and hepatocarcinogenic in rats. The kinetics of furan biotransformation by male F-344 rats were studied in vivo and in vitro in order to understand target tissue dosimetry. A physiologically based pharmacokinetic (PBPK) model for furan in rats was developed from gas uptake studies using initial furan concentrations of 100, 500, 1050, and 3850 ppm. Tissue partition coefficients for furan were determined in vitro using vial equilibration techniques. Furan gas uptake kinetics in vivo were described by a single saturable process with a Vmax of 27.0 mumol/hr/250 g rat and a KM of 2.0 microM. Furan metabolism in vivo was inhibited by pyrazole. The furan PBPK model adequately simulated blood and liver furan concentrations following 4-hr inhalation exposures to 52, 107, and 208 ppm furan. The biotransformation of furan was studied in freshly isolated rat hepatocytes in vitro and compared to biotransformation in vivo. Furan biotransformation by isolated rat hepatocytes exhibited a KM of 0.4 microM and a Vmax of 0.018 mumol/hr/10(6) cells. Inhibition and induction studies indicated that cytochrome P450 was the catalyst of furan oxidation. Acetone pretreatment of the rats produced a five-fold increase in the rate of the hepatocyte oxidation of furan, suggesting an important role for cytochrome P450 2E1. The Vmax determined in hepatocytes in vitro extrapolated to 23.0 mumol/hr/250 g rat, assuming 128 x 10(6) hepatocytes/g liver. Incorporation of the in vitro hepatocyte kinetic parameters into the PBPK model for furan accurately simulated in vivo pharmacokinetics. These results suggest that freshly isolated hepatocytes are a valuable in vitro system for predicting chemical pharmacokinetics in vivo.
The in vitro metabolism of four s-triazine herbicides (atrazine, terbuthylazine, ametryne, and terbutryne) was studied using liver microsomes from rats, pigs, and humans. New HPLC methods with UV detection were developed for the analyses of the incubations. Principal phase I reactions were N-monodealkylation, hydroxylation of the isopropyl or tert-butyl moiety, and sulfoxidation of the substrates in all species. Bidealkylation, 2-hydroxylation, or cleavage of the tert-butyl moiety could not be found in this system. The sulfoxidation of the 2-methylthio-s-triazines exceeded catalysis of the other metabolic reactions by 3-4-fold in all species. In general, all species produced the same types of metabolites, but with species-specific differences in the ratios of the metabolites. Species-specific stereoselective formation of a new chiral isopropyl-hydroxylated metabolite from atrazine was investigated using chiral HPLC techniques. The stereoselective production of this metabolite was different in the different species, with S/R ratios of 76:24 in rats, 49:51 in pigs, and 28:72 in humans.
We studied atrazine (ATZ) metabolism in male and female rat liver microsomes in vitro, and the major metabolite was deisopropylatrazine (DeiPr-ATZ) with deethylatrazine (DeEt-ATZ) and 1-hydroxyisopropylatrazine (iPrOH-ATZ) as minor metabolites in both sexes. The enzyme kinetics of ATZ biotransformation were examined by means of Eadie-Hofstee analyses. Although no remarkable sex difference of Michaelis Menten values for each pathway was observed, Cl(int)S (Vmax/Km) for DeiPr-ATZ, DeEt-ATZ and iPrOH-ATZ were slightly higher in female than in male rats. The formation of DeiPr-ATZ, DeEt-ATZ and iPrOH-ATZ from ATZ was substantially inhibited by SKF-525A, metyrapone, diallyl sulfide, 7-ethoxycoumarin, benzphetamine, nicotine, testosterone and lauric acid in both sexes. Cimetidine effectively inhibited the formation of all metabolites in male rats. On the other hand, the inhibition rates of the formation of DeiPr-ATZ and iPrOH-ATZ by cimetidine in female rats were lower than those in male rats, and DeEt-ATZ was hardly affected by the chemicals. In contrast with the results for cimetidine, the inhibition of ATZ biotransformation by bufuralol was more effective in female than in male rats. Anti-rat CYP2B1 and CYP2E1 antibodies effectively inhibited DeiPr-ATZ, DeEt-ATZ and iPrOH-ATZ formations in both sexes. Anti-rat CYP2C11 antibody also inhibited the three metabolites in both sexes, with the inhibition rates higher in male than in female rats, similar to cimetidine. In the case of anti-rat CYP2D1 antibody, the inhibitory effect on ATZ biotransformation in male rats was less than that in female rats. On the other hand, anti-rat CYP1A2, CYP3A2 and CYP4A1 antibodies did not affect the ATZ biotransformation in either sex. There was no significant correlation between the formation rate of ATZ metabolites and P450 isoform levels in either sex. These results may mean that CYP2B2, CYP2C11, CYP2D1 (only iPrOH-ATZ formation) and CYP2E1 in male rats, and CYP2B2, CYP2D1 and CYP2E1 in female rats are involved ATZ metabolism in liver, and that the substrate specificity of P450 isoforms for ATZ is broad.
The in vitro metabolism of chlorotriazines, simazine (SIZ), atrazine (ATZ), and propazine (PRZ) was studied using control, 3-methylcholanthrene-, phenobarbital-, pyridine-, dexamethasone-, and clofibrate-treated rat liver microsomes. The metabolites were determined by HPLC. The principal reactions by cytochrome P450 (P450) system were N-monodealkylation and isopropylhydroxylation in all rat liver microsomes. As a result, 2-chloro-4-ethylamino-6-amino-1,3,5-triazine (M1) (SIZ-M1 for SIZ and ATZ-M1 for ATZ) and 2-chloro-4-amino-6-isopropylamino-1,3, 5-triazine (M2) (ATZ-M2 for ATZ and PRZ-M2 for PRZ), 2-chloro-4-ethylamino-6-(1-hydroxyisopropylamino)-1,3,5-triazine (M3) (ATZ-M3 for ATZ), and 2-chloro-4-isopropylamino-6-(1-hydroxyisopropylamino)-1,3,5-triazi ne (M4) (PRZ-M4 for PRZ) were detected as the metabolites. N-bidealkylation and 2-hydroxylation were not found in this system. The formation rates of SIZ-M1, ATZ-M1, ATZ-M2, and PRZ-M2 were markedly induced by 3-methylcholanthrene, phenobarbital, and pyridine. On the other hand, the formation rates of ATZ-M3 and PRZ-M4 were significantly induced by phenobarbital, pyridine, and/or clofibrate, but not by 3-methylcholanthrene. The enzyme kinetics of chlorotriazine metabolism were examined by mean of Eadie-Hofstee analyses. Although there was no remarkable difference of Km for the products in chlorotriazine metabolism among the microsomes tested, the Vmax and Clint (Vmax/Km) for the products in chlorotriazine metabolism are affected by P450 inducers, except for dexamethasone. The formation rates of SIZ-M1, ATZ-M1, ATZ-M2, and PRZ-M2 were significantly correlated with 7-ethoxyresorufin O-deethylase, acetanilide 4-hydroxylase, 7-ethoxycoumarin O-deethylase, 4-nitrophenol 2-hydroxylase, and testosterone 7alpha-hydroxylase activities and CYP1A1/2 level, whereas the formation rates of ATZ-M3 and PRZ-M4 were significantly correlated with testosterone 16beta-hydroxylase, bufuralol 1'-hydroxylase, and 4-nitrophenol 2-hydroxylase activities and CYP2B1/2 level. These results suggest that the inducibility in metabolism of SIZ, ATZ, and PRZ is different between N-monodealkylation and isopropylhydroxylation and that the N-monodealkylation and isopropylhydroxylation are induced by CYP1A1/2, CYP2B1/2, and CYP2B1/2, respectively.
1. The in vitro metabolism of chlorotriazines, simazine (SIZ), atrazine (ATZ) and propazine (PRZ) in liver microsomes from rat, mouse and guinea pig and the oestrogenic activity of chlorotriazines and their main metabolites have been studied. 2. The formation rates of products in chlorotriazine metabolism were determined by HPLC. The principal reactions catalysed by the cytochrome P450 (P450) system were N-monodealkylation and isopropylhydroxylation in all liver microsomes. As a result, 2-chloro-4-ethylamino-6-amino-1,3,5-triazine (M1) (SIZ-M1 for SIZ and ATZ-M1 for ATZ) and 2-chloro-4-amino-6-isopropylamino-1,3,5-triazine (M2) (ATZ-M2 for ATZ and PRZ-M2 for PRZ), and 2-chloro-4-ethylamino-6-(1-hydroxyisopropylamino)-1,3,5-triazine (M3) (ATZ-M3 for ATZ) and 2-chloro-4-isopropylamino-6-(1-hydroxyisopropylamino)-1,3,5-triazi ne (M4) (PRZ-M4 for PRZ) were detected as the metabolites. N-bidealkylation was not found in this system. 3. The formation rates of N-deethylated metabolites (SIZ-M1 and ATZ-M2) were generally higher in mouse than in rat and guinea pig. The formation rates of N-deisopropylated metabolites (ATZ-M1 and PRZ-M2) in guinea pig were the lowest among the three animal species. The formation rates of isopropylhydroxylated metabolites (ATZ-M3 and PRZ-M4) were remarkably low in mouse compared with rat and guinea pig. 4. The enzyme kinetics of chlorotriazine metabolism were examined by Eadie-Hofstee analyses. Some species differences in Michaelis-Menten parameters for each metabolite were observed, and the ranking orders were varied among the metabolites. 5. The binding affinity of chlorotriazines (SIZ, ATZ and PRZ) and their metabolites (M1-4) for recombinant human oestrogen receptor-alpha was assayed using the fluorescence polarization method. The binding affinity of M2 was significantly higher than those of parent compounds and other metabolites, although the oestrogenic activity was remarkably low compared with that of 17beta-oestradiol (E2). 6. These results suggest that the pattern of metabolism of SIZ, ATZ and PRZ by the P450 system differs extensively among rat, mouse and guinea pig, and that M2 may be an activated metabolite of chlorotriazines.
Benzene (C6H6) is a highly flammable, colorless liquid. Ubiquitous exposures result from its presence in gasoline vapors, cigarette smoke, and industrial processes. Benzene increases the incidence of leukemia in humans when they are exposed to high doses for extended periods; however, leukemia risks in humans at low exposures are uncertain. The exposure-dose-response relationship of benzene in humans is expected to be nonlinear because benzene undergoes a series of metabolic transformations, detoxifying and activating, in the liver, resulting in multiple metabolites that exert toxic effects on the bone marrow. We developed a physiologically based pharmacokinetic model for the uptake and elimination of benzene in mice to relate the concentration of inhaled and orally administered benzene to the tissue doses of benzene and its key metabolites, benzene oxide, phe nol, and hydroquinone. As many parameter values as possible were taken from the literature; in particular, metabolic parameters obtained from in vitro studies with mouse liver were used since comparable parameters are also available for humans. Parameters estimated by fitting the model to published data were first-order rate constants for pathways lacking in vitro data and the concentrations of microsomal and cytosolic protein, which effectively alter overall enzyme activity. The model was constrained by using the in vitro metabolic parameters (maximum velocities, first-order rate constants, and saturation parameters), and data from multiple laboratories and experiments were used. Despite these constraints and sources of variability, the model simulations matched the data reasonably well in most cases, showing that in vitro metabolic constants can be successfully extrapolated to predict in vivo data for benzene metabolism and dosimetry. Therefore in vitro metabolic constants for humans can subsequently be extrapolated to predict the dosimetry of benzene and its metabolites in humans. This will allow us to better estimate the risks of adverse effects from low-level benzene exposures.
The ability of various human derived in vitro systems to predict various aspects of the in vivo metabolism and kinetics of almokalant have been investigated in a multicenter collaborative study. Although almokalant has been withdrawn from further clinical development, its metabolic and pharmacokinetic properties have been well characterized. Studies with precision-cut liver slices, primary hepatocyte cultures, and hepatic microsomal fractions fortified with UDP-glucuronic acid all suggested that almokalant is mainly glucuronidated to the stereoisomers M18a and M18b, which is in good agreement with the results in vivo. Both in vivo and in vitro studies indicate that the formation of M18b dominates over that of M18a, although the difference is more pronounced with the in vitro systems. Molecular modeling, cDNA-expressed enzyme analysis, correlation analysis, and inhibition studies did not clearly indicate which P450 enzymes catalyze the oxidative pathways, which may indicate a problem in identifying responsible enzymes for minor metabolic routes by in vitro methods. All of the in vitro systems underpredicted the metabolic clearance of almokalant, which has previously been reported to be a general problem for drugs that are cleared by P450-dependent metabolism. Although few studies on in vivo prediction of primarily glucuronidated drugs have appeared, in vitro models may consistently underpredict in vivo metabolic clearance. We conclude that in vitro systems, which monitor phase II metabolism, would be beneficial for prediction of the in vivo metabolism, although all of the candidate liver-derived systems studied here, within their intrinsic limitations, provided useful information for predicting metabolic routes and rates.
A physiological pharmacokinetic (PPK) model, with blood, body, and brain compartments, was developed to estimate total plasma chlorotriazine (CI-TRI) time courses (i.e., atrazine [ATRA] and its three chlorinated metabolites) after oral dosing with ATRA. The model, based on disposition data for 14C-ATRA, tracked two pools of compounds: (1) ATRA and chlorinated metabolites (i.e., the CI-TRIs) and (2) glutathione conjugates. The PPK model developed from total radioactivity was valuable for assessing total plasma CI-TRI concentrations, estimating blood protein binding rates of CI-TRIs, and inferring relationships between tissue exposures of CI-TRIs and administered dose. Absorption of radioactivity into plasma was slow with a rate constant of 0.2 h-1. 14C-disposition data indicated that CI-TRIs react with red blood cells (presumably hemoglobin) and plasma proteins. Second-order rates of reaction of CI-TRIs with hemoglobin and plasma protein were estimated to be 0.008 L/mmol/h and 1.14 x 10(-7) L/mg/h, respectively. A time-course study, conducted as part of this study, evaluated the absorption, disposition, and elimination characteristics of individual CI-TRIs in plasma after a single oral dose of 90 mg ATRA/kg and indicated (1) that slow uptake into blood reflected both absorption and slow dissolution of the ATRA slurry and (2) that diaminochloro-s-triazine (DACT) was the major, persistent plasma CI-TRI after oral dosing. Optimally, PK model development for pesticide compounds like atrazine should include a combination of radiolabeled studies for residues and speciation studies of important metabolites.
Comparative Metabolism of Atrazine by Mammalian Hepatocytes: Progress Report. Ciba-Geigy Corporation
- B Thede
Thede, B., 1988. Comparative Metabolism of Atrazine by Mammalian
Hepatocytes: Progress Report. Ciba-Geigy Corporation, Greensboro,
North Carolina.
6 mM) were prepared in acetone. DACT was prepared in Fig. 1. Schematic of the P450 mediated oxidative metabolism of (a) atrazine (ATRA) to (b) 2-chloro-4-ethylamino-6-amino-1,3,5-triazine (ETHYL) and (c) 2-chloro-4-amino-6-isopropylamino-1
- Standards
- Atra
- Ethyl
- Iso
- Cya
Standards of ATRA, ETHYL, ISO, and CYA (all at 4.6 mM) were prepared in acetone. DACT was prepared in Fig. 1. Schematic of the P450 mediated oxidative metabolism of (a) atrazine (ATRA) to (b) 2-chloro-4-ethylamino-6-amino-1,3,5-triazine (ETHYL) and (c) 2-chloro-4-amino-6-isopropylamino-1,3,5-triazine(ISO), and subsequent metabolism to (d) diaminochlorotriazine (DACT).
Study of 14 C-Atrazine Dose/Response Relationship in the Rat
- B Thede
Thede, B., 1987. Study of 14 C-Atrazine Dose/Response Relationship in
the Rat. Ciba-Geigy Corporation, Greensboro, North Carolina, pp. 133.
Effect of 6 months of atrazine or hydroxyatrazine on the leutinizing hormone surge in female Sprague-Dawley and Fischer 344 rats
- J C Eldridge
- D Minnema
- C B Breckenridge
- J E Mcfarland
- J T Stevens
Eldridge, J.C., Minnema, D., Breckenridge, C.B., McFarland, J.E.,
Stevens, J.T., 2001. Effect of 6 months of atrazine or hydroxyatrazine
on the leutinizing hormone surge in female Sprague-Dawley and
Fischer 344 rats. The Toxicologist.
Effect of 6 months of atrazine or hydroxyatrazine on the leutinizing hormone surge in female Sprague–Dawley and Fischer 344 rats
- Eldridge