Matthew K. Ross

Identification of Rat and Human Cytochrome P450 Isoforms and a Rat Serum Esterase That Metabolize the Pyrethroid Insecticides Deltamethrin and Esfenvalerate

The metabolism of (αS)-cyano-3-phenoxybenzyl (1R, 3R)-cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane carboxylate (deltamethrin) and (αS)-cyano-3-phenoxybenzyl 2-(4-chlorophenyl)-3-methylbutyrate (esfenvalerate) by rat and human liver microsomes differs with respect to the biotransformation pathway (oxidation versus hydrolysis) responsible for their clearance. This study aims to further explore the species differences in the metabolism of these chemicals. Using a parent depletion approach, rat and human cytochromes P450 (P450s) were screened for their ability to eliminate deltamethrin or esfenvalerate during in vitro incubations. Rat P450 isoforms CYP1A1, CYP2C6, CYP2C11, and CYP3A2 and human P450 isoforms CYP2C8, CYP2C19, and CYP3A5 were capable of metabolizing either pyrethroid. Human CYP2C9 metabolized esfenvalerate but not deltamethrin. Rat and human P450s that metabolize esfenvalerate and deltamethrin do so with similar kinetics. In addition to the liver, a potential site of metabolic elimination of pyrethroids is the blood via serum carboxylesterase (CE) hydrolysis. The serum of rats, but not humans, contains significant quantities of CE. Deltamethrin and esfenvalerate were metabolized effectively by rat serum and a purified rat serum CE. In contrast, neither pyrethroid was metabolized by human serum or purified human serum esterases (acetylcholinesterase and butyrylcholinesterase). These studies suggest that the difference in rates of oxidative metabolism of pyrethroids by rat and human hepatic microsomes is dependent on the expression levels of individual P450 isoforms rather than their specific activity. Furthermore, these studies show that the metabolic elimination of deltamethrin and esfenvalerate in blood may be important to their disposition in rats but not in humans.

Species Differences in the in Vitro Metabolism of Deltamethrin and Esfenvalerate: Differential Oxidative and Hydrolytic Metabolism by Humans and Rats

Pyrethroids are neurotoxic pesticides whose pharmacokinetic behavior plays a role in their potency. This study examined the elimination of esfenvalerate and deltamethrin from rat and human liver microsomes. A parent depletion approach in the presence and absence of NADPH was used to assess species differences in biotransformation pathways, rates of elimination, and intrinsic hepatic clearance. Esfenvalerate was eliminated primarily via NADPH-dependent oxidative metabolism in both rat and human liver microsomes. The intrinsic hepatic clearance (CLINT) of esfenvalerate was estimated to be 3-fold greater in rodents than in humans on a per kilogram body weight basis. Deltamethrin was also eliminated primarily via NADPH-dependent oxidative metabolism in rat liver microsomes; however, in human liver microsomes, deltamethrin was eliminated almost entirely via NADPH-independent hydrolytic metabolism. The CLINT for deltamethrin was estimated to be 2-fold more rapid in humans than in rats on a per kilogram body weight basis. Metabolism by purified rat and human carboxylesterases (CEs) were used to further examine the species differences in hydrolysis of deltamethrin and esfenvalerate. Results of CE metabolism revealed that human carboxylesterase 1 (hCE-1) was markedly more active toward deltamethrin than the class 1 rat CEs hydrolase A and B and the class 2 human CE (hCE-2); however, hydrolase A metabolized esfenvalerate 2-fold faster than hCE-1, whereas hydrolase B and hCE-1 hydrolyzed esfenvalerate at equal rates. These studies demonstrate a significant species difference in the in vitro pathways of biotransformation of deltamethrin in rat and human liver microsomes, which is due in part to differences in the intrinsic activities of rat and human carboxylestersases.

Disposition of the Herbicide 2-Chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine (Atrazine) and Its Major Metabolites in Mice: A Liquid Chromatography/Mass Spectrometry Analysis of Urine, Plasma, and Tissue Levels

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 ATRs 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 μM) at 4 h (5–125 mg/kg) and 1 h (250 mg/kg) after dosing, but levels quickly declined to <0.1 μM by 12 h in all the groups. In contrast, strikingly high concentrations of DACT (1.5–50 μM), 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.

Analysis of in vivo and in vitro DNA strand breaks from trihalomethane exposure.

Background Epidemiological studies have linked the consumption of chlorinated surface waters to an increased risk of two major causes of human mortality, colorectal and bladder cancer. Trihalomethanes (THMs) are by-products formed when chlorine is used to disinfect drinking water. The purpose of this study was to examine the ability of the THMs, trichloromethane (TCM), bromodichloromethane (BDCM), dibromochloromethane (DBCM), and tribromomethane (TBM), to induce DNA strand breaks (SB) in (1) CCRF-CEM human lymphoblastic leukemia cells, (2) primary rat hepatocytes (PRH) exposed in vitro, and (3) rats exposed by gavage or drinking water. Methods DNA SB were measured by the DNA alkaline unwinding assay (DAUA). CCRF-CEM cells were exposed to individual THMs for 2 hr. Half of the cells were immediately analyzed for DNA SB and half were transferred into fresh culture medium and incubated for an additional 22 hr before testing for DNA SB. PRH were exposed to individual THMs for 4 hr then assayed for DNA SB. F344/N rats were exposed to individual THMs for 4 hr, 2 weeks, and to BDCM for 5 wk then tested for DNA SB. Results CCRF-CEM cells exposed to 5- or 10-mM brominated THMs for 2 hr produced DNA SB. The order of activity was TBM>DBCM>BDCM; TCM was inactive. Following a 22-hr recovery period, all groups had fewer SB except 10-mM DBCM and 1-mM TBM. CCRF-CEM cells were found to be positive for the GSTT1-1 gene, however no activity was detected. No DNA SB, unassociated with cytotoxicity, were observed in PRH or F344/N rats exposed to individual THMs. Conclusion CCRF-CEM cells exposed to the brominated THMs at 5 or 10 mM for 2 hr showed a significant increase in DNA SB when compared to control cells. Additionally, CCRF-CEM cells exposed to DBCM and TBM appeared to have compromised DNA repair capacity as demonstrated by an increased amount of DNA SB at 22 hr following exposure. CCRF-CEM cells were found to be positive for the GSTT1-1 gene, however no activity was detected. No DNA SB were observed in PRH or F344/N rats exposed to individual THMs.

Evaluation of the ‘side door’ in carboxylesterase-mediated catalysis and inhibition

Abstract Structures of mammalian carboxylesterases (CEs) reveal the presence of a ‘side door’ that is proposed to act as an alternative pore for the trafficking of substrates and products. p-Nitrobenzyl esterase (pnb CE) from Bacillus subtilis exhibits close structural homology and a similar side-door domain as mammalian CEs. We investigated the role of a specific ‘gate’ residue at the side door (i.e., Leu 362) during pnb CE-catalyzed hydrolysis of model esters, pesticides, and lipids. Recombinant pnb CE proteins containing mutations at position 362 demonstrated markedly lower k cat and k cat/K m values. The mutation with the most significant impact on catalysis was the L362R mutant (k cat/K m was 22-fold lower). Moreover, the ability of the L362R mutant to be inhibited by organophosphates (OP) was also lower. Investigation into the altered catalytic proficiency using pH-activity studies indicated that the catalytic triad of the mutant enzyme was preserved. Furthermore, viscosity variation and carbamate inhibition experiments indicated that rates of substrate association and acylation/deacylation were lower. Finally, recombinant CEs were found to possess lipolytic activity toward cholesteryl oleate and 2-arachidonylglycerol. In summary, the L362R mutant CE markedly slowed the rate of ester hydrolysis and was less sensitive to OP inhibition. The apparent causes of the diminished catalysis are discussed.

Effects of Toxicologically Relevant Xenobiotics and the Lipid-Derived Electrophile 4-Hydroxynonenal on Macrophage Cholesterol Efflux: Silencing Carboxylesterase 1 Has Paradoxical Effects on Cholesterol Uptake and Efflux

Cholesterol cycles between free cholesterol (unesterified) found predominantly in membranes and cholesteryl esters (CEs) stored in cytoplasmic lipid droplets. Only free cholesterol is effluxed from macrophages via ATP-binding cassette (ABC) transporters to extracellular acceptors. Carboxylesterase 1 (CES1), proposed to hydrolyze CEs, is inactivated by oxon metabolites of organophosphorus pesticides and by the lipid electrophile 4-hydroxynonenal (HNE). We assessed the ability of these compounds to reduce cholesterol efflux from foam cells. Human THP-1 macrophages were loaded with [3H]-cholesterol/acetylated LDL and then allowed to equilibrate to enable [3H]-cholesterol to distribute into its various cellular pools. The cholesterol-engorged cells were then treated with toxicants in the absence of cholesterol acceptors for 24 h, followed by a 24 h efflux period in the presence of toxicant. A concentration-dependent reduction in [3H]-cholesterol efflux via ABCA1 (up to 50%) was found for paraoxon (0.1–10 μM), whereas treatment with HNE had no effect. A modest reduction in [3H]-cholesterol efflux via ABCG1 (25%) was found after treatment with either paraoxon or chlorpyrifos oxon (10 μM each) but not HNE. No difference in efflux rates was found after treatments with either paraoxon or HNE when the universal cholesterol acceptor 10% (v/v) fetal bovine serum was used. When the re-esterification arm of the CE cycle was disabled in foam cells, paraoxon treatment increased CE levels, suggesting the neutral CE hydrolysis arm of the cycle had been inhibited by the toxicant. However, paraoxon also partially inhibited lysosomal acid lipase, which generates cholesterol for efflux, and reduced the expression of ABCA1 protein. Paradoxically, silencing CES1 expression in macrophages did not affect the percent of [3H]-cholesterol efflux. However, CES1 mRNA knockdown markedly reduced cholesterol uptake by macrophages, with SR-A and CD36 mRNA reduced 3- and 4-fold, respectively. Immunoblots confirmed SR-A and CD36 protein downregulation. Together, these results suggest that toxicants, e.g., oxons, may interfere with macrophage cholesterol homeostasis/metabolism.

Exposure to p,p'-DDE Alters Macrophage Reactivity and Increases Macrophage Numbers in Adipose Stromal Vascular Fraction.

Exposure to p,p-DDE (DDE), the main bioaccumulative metabolite of the organochlorine insecticide p,p-DDT, is associated with a higher prevalence of obesity, dyslipidemia, insulin resistance, metabolic syndrome, and immunomodulation. The present study was carried out to determine whether DDE perturbs adipose tissue homeostasis through modulation of macrophage function. Treatment with DDE or a cyclooxygenase-2 inhibitor prior to lipopolysaccharide exposure significantly decreased production of prostaglandins (PG) from J774a.1 macrophages inxa0vitro. Similarly, J774A.1 cell lysates incubated with DDE or a specific cyclooxygenase-2 inhibitor (NS-398) produced significantly less PGE2 and PGF2α. Macrophage polarization studies revealed a pattern of DDE effects that were not fully consistent with a purely pro- or purely anti- M1 or M2 effect. However, DDE suppressed expression of two M1 markers (induced by an M1 stimulus) and enhanced expression of an M2 marker (induced by an M2 stimulus). Further studies including assessment of macrophage function are needed to fully characterize the effects of DDE on macrophage polarization. Obesity is characterized by an increase in the number of resident adipose tissue macrophages. To assess monocyte/macrophage recruitment to the adipose tissue inxa0vivo, male C57Bl/6H mice were treated with 2u2009mg/kg DDE or corn oil vehicle for 5 days by gavage. Epididymal fat pads were digested and macrophage populations were analyzed by flow cytometry. In DDE-treated animals, there was a significant increase (37%) in F4/80(+)CD11b(+) macrophages/g of epididymal adipose over vehicle (Pu2009<u2009.05). Together, these results suggest a role for DDE in the enhancement of adipose tissue macrophage recruitment and/or proliferation, as well as modulation of immune cell function that may contribute to the etiology of metabolic diseases associated with organochlorine exposure.

[35S]-Labeling of the Salmonella typhimurium glutathione pool to assess glutathione-mediated DNA binding by 1,2-dibromoethane

Biotransformation of drugs and environmental chemicals to reactive intermediates is often studied with the use of radiolabeled compounds that are synthesized by expensive and technically difficult procedures. In general, glutathione (GSH) conjugation serves as a detoxification mechanism, and conjugation of reactive intermediates with GSH is often a surrogate marker of reactive species formation. However, several halogenated alkanes can be bioactivated by GSH to yield highly reactive GSH conjugates, some of which are DNA-reactive (e.g. conjugates of 1,2-dibromoethane). The purpose of this study was to metabolically radiolabel the in vivo GSH pool of Salmonella typhimurium with a [35S]-label and to examine the GSH-mediated bioactivation of a model haloalkane, 1,2-dibromoethane, by measuring the binding of [35S]-label to DNA. The strain of Salmonella used in this study had been transformed previously with the gene that codes for rat glutathione transferase theta 1-1 (GSTT1-1), an enzyme that can catalyze formation of genotoxic GSH conjugates. Bacteria were grown to mid-log phase and then incubated with [35S]-L-cysteine in minimal medium (thio-free) until stationary phase of growth was reached. At this stage, the specific activity of Salmonella GSH was estimated to be 7.1 mCi/mmol by derivatization and subsequent HPLC analysis, and GSTT1-1 enzyme activity was still demonstrable in Salmonella cytosol following growth in a minimal medium. The [35S]-labeled bacteria were then exposed to 1,2-dibromoethane (1 mM), and the Salmonella DNA was subsequently purified to quantify [35S]-binding to DNA. The amount of [35S]-label that was covalently bound to DNA in the GSTT1-1-expressing Salmonella strain (33.2 nmol/mg DNA) was sevenfold greater than that of the control strain that does not express GSTT1-1. Neutral thermal hydrolysis of the DNA yielded a single [35S]-labeled adduct with a similar t(R) as S-[2-(N(7)-guanyl)ethyl]GSH, following HPLC analysis of the hydrolysate. This adduct accounted for 95% of the total [35S]-label bound to DNA. Thus, this [35S]-radiolabeling protocol may prove useful for studying the DNA reactivity of GSH conjugates of other halogenated alkanes in a cellular context that maintains GSH at normal physiological levels. This is also, to our knowledge, the first demonstration of de novo incorporation of [35S]-L-cysteine into the bacterial GSH pool.