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Semiconductor quantum dots (Qdots) are a promising new technology with benefits in the areas of medical diagnostics and therapeutics. Qdots generally consist of a semiconductor core, capping shell, and surface coating. The semiconductor core of Qdots is often composed of group II and VI metals (e.g., Cd, Se, Te, Hg) that are known to have toxic properties. Various surface coatings have been shown to stabilize Qdots and thus shield cells from the toxic properties of their core elements. In this study, HepG2 cells and primary human liver (PHL) cells were chosen as in vitrotissue culture models of human liver to examine the possible adverse effects of tri-n-octylphosphine oxide, poly(maleic anhydride-alt-1-tetradecene) copolymer (TOPO-PMAT)-coated CdSe/ZnS Qdots (TOPO-PMAT Qdots). The TOPO-PMAT coating is desirable for increasing aqueous solubility and ease of conjugation to targeting moieties (e.g., aptamers and peptides). HepG2 cells avidly incorporated these TOPO-PMAT Qdots into subcellular vesicles. However, PHL cells did not efficiently take up TOPO-PMAT Qdots, but nonparenchymal cells did (especially Kupffer cells). No acute toxicity or morphological changes were noted in either system at the exposure levels used (up to 40 nM). However, cellular stress markers and pro-inflammatory cytokines/chemokines were increased in the PHL cell cultures, suggesting that TOPO-PMAT Qdots are not likely to cause acute cytotoxicity in the liver but may elicit inflammation/hepatitis, demonstrating the importance of relevant preclinical safety models. Thus, further in vivostudies are warranted to ensure that TOPO-PMAT-coated Qdots used in biomedical applications do not induce inflammatory responses as a consequence of hepatic uptake.

Parkinson disease (PD) typically affects the cortical regions during the later stages of disease, with neuronal loss, gliosis, and formation of diffuse cortical Lewy bodies in a significant portion of patients with dementia. To identify novel proteins involved in PD progression, we prepared synaptosomal fractions from the frontal cortices of pathologically verified PD patients at different stages along with age-matched controls. Protein expression profiles were compared using a robust quantitative proteomic technique. Approximately 100 proteins displayed significant differences in their relative abundances between PD patients at various stages and controls; three of these proteins were validated using independent techniques. One of the confirmed proteins, glutathione S-transferase Pi, was further investigated in cellular models of PD, demonstrating that its level was intimately associated with several critical cellular processes that are directly related to neurodegeneration in PD. These results have, for the first time, suggested that the levels of glutathione S-transferase Pi may play an important role in modulating the progression of PD.

Sulforaphane (SFN) is a biologically active phytochemical found abundantly in broccoli. SFN has been promoted as a putative chemopreventive agent to reduce cancer, and most studies have associated its anti-cancer effects with the induction of phase II xenobiotic metabolism enzymes via activation of the Keap1/Nrf2 antioxidant response pathway. Interestingly, SFN can significantly down-regulate cytochrome P450 3A4 (CYP3A4) expression in human primary hepatocytes. CYP3A4 is responsible for the hepatic and intestinal metabolism of numerous protoxicants, pharmaceutical compounds, and endogenous sterols. Among the most important mediators of CYP3A4 expression is the nuclear hormone receptor, steroid and xenobiotic receptor (SXR; also called "hPXR"). SXR functions as a xenobiotic sensor to coordinately regulate xenobiotic metabolism via transcriptional regulation of xenobiotic-detoxifying enzymes and transporters. Here, we report that SFN is a specific antagonist of human SXR and that it inhibits SXR-mediated induction of drug clearance. SFN can bind directly to SXR, inhibit SXR coactivator recruitment, and efficiently repress SXR activities. Furthermore, SFN inhibited SXR-mediated CYP3A4 expression and CYP3A4-catalyzed midazolam clearance in human primary hepatocytes. Thus, SFN is the first identified naturally occurring antagonist for SXR (hPXR). Because induction of CYP3A4 can result in adverse drug responses (e.g., lack of efficacy), which are a major public health problem, this discovery could lead to the development of important new therapeutic and dietary approaches to reduce the frequency of undesirable inducer-drug interactions.

Aflatoxin B1 (AFB1) is a potent human hepatotoxin and hepatocarcinogen produced by the mold Aspergillus flavus. In human, AFB1 is bioactivated by cytochrome P450 (CYP450) enzymes, primarily CYP1A2, to the genotoxic epoxide that forms N7-guanine DNA adducts. To characterize the transcriptional responses to genotoxic insults from AFB1, a strain of Saccharomyces cerevisiae engineered to express human CYP1A2 was exposed to doses of AFB1 that resulted in minimal lethality, but substantial genotoxicity. Flow cytometric analysis demonstrated a dose and time dependent S phase delay under the same treatment conditions, indicating a checkpoint response to DNA damage. Replicate cDNA microarray analyses of AFB1 treated cells showed that about 200 genes were significantly affected by the exposure. The genes activated by AFB1-treatment included RAD51, DUN1 and other members of the DNA damage response signature reported in a previous study with methylmethane sulfonate and ionizing radiation [A.P. Gasch, M. Huang, S. Metzner, D. Botstein, S.J. Elledge, P.O. Brown, Genomic expression responses to DNA-damaging agents and the regulatory role of the yeast ATR homolog Mec1p, Mol. Biol. Cell 12 (2001) 2987–3003]. However, unlike previous studies using highly cytotoxic doses, environmental stress response genes [A.P. Gasch, P.T. Spellman, C.M. Kao, O. Carmel-Harel, M.B. Eisen, G. Storz, D. Botstein, P.O. Brown, Genomic expression programs in the response of yeast cells to environmental changes, Mol. Biol. Cell 11 (2000) 4241–4257] were largely unaffected by our dosing regimen. About half of the transcripts affected are also known to be cell cycle regulated. The most strongly repressed transcripts were those encoding the histone genes and a group of genes that are cell cycle regulated and peak in M phase and early G1. These include most of the known daughter-specific genes. The rapid and coordinated repression of histones and M/G1-specific transcripts cannot be explained by cell cycle arrest, and suggested that there are additional signaling pathways that directly repress these genes in cells under genotoxic stress.

Cyclohexanone monooxygenase (CMO) is a soluble flavoenzyme originally isolated from Acinetobacterspp. which carries out Baeyer-Villiger reactions with cyclic ketone substrates. In the present study we cloned the AcinetobacterCMO gene and modified it for facile purification from heterologous expression systems by incorporation of a His6-tag at its C-terminus. A single purification step employing metal (Ni2+)-affinity column chromatography provided essentially homogeneous enzyme in yields of 69–72%. The properties of the purified, recombinant enzymes (rCMO) were compared with that of native CMO (nCMO) isolated from Acinetobactercultures grown in the presence of cyclohexanone. The specific activities of His6-tagged rCMO and nCMO toward their index substrate, cyclohexanone, were similar and ranged from 14 to 20 μmol/min/mg. nCMO and rCMO from the Escherichia coliexpression system exhibited molecular masses, determined by electrospray mass spectrometry, of 60,800 and 61,615 Da, respectively, an increase for the recombinant enzyme equivalent to the mass of the His6-tag. However, rCMO expressed in Saccharomyces cerevisiaeconsistently exhibited a mass some 50 Da larger than rCMO expressed in bacteria. Edman degradation confirmed that rCMO purified from the E. colisystem and nCMO shared the same N-terminal sequence, whereas no sequence information could be obtained for rCMO expressed in yeast. Therefore, the yeast-expressed enzyme possesses an additional posttranslational modification(s), possibly acylation, at the N-terminus. Expression in E. coliis the preferred system for future site-directed mutagenesis studies and crystallization efforts.

Acetaminophen (APAP) is known to cause centrilobular hepatic necrosis under overdose conditions. This is thought to be mediated via the P450-generated reactive intermediateN-acetyl-p-benzoquinone imine (NAPQI). Initially, NAPQI is detoxified by conjugation with glutathione (GSH), but once GSH is depleted, NAPQI reacts more extensively with hepatic proteins leading to hepatocellular damage. The P450 isoforms thought to be responsible for APAP hepatotoxicity in humans are CYP2E1, CYP1A2, and CYP3A4, and thus, we have investigated the effect of murine Cyp1a2 on APAP hepatotoxicity using Cyp1a2 knockout mice (Lianget al., Proc. Natl. Acad. Sci. USA93, 1671–1676, 1996). Doses of 250 mg/kg were markedly hepatotoxic in these mice, and surprisingly, deaths only occurred in the knock-out and heterozygote mice over a 24-h period after dosing. Furthermore, there were no significant differences among survivors of any genotype in serum ALT concentrations, a well correlated indicator of APAP hepatotoxicity in mice. Finally, no differences were observed in the urinary metabolites excreted over the 24-h period, including those derived from GSH conjugation of the major reactive metabolite NAPQI. Consistent with the effects on hepatotoxicity and metabolism, 2 h after hepatotoxic doses (500 mg/kg, ip) of APAP no significant differences were observed in total whole liver homogenate nonprotein thiol concentrations among the three genotypes even though hepatic thiols were decreased compared to control animals (>90%). In addition, when the liver cytosol and microsome samples were examined by immunoblotting for the presence of APAP-protein adducts using a specific antiserum, there were no observable differences in either the intensity of staining or in the spectrum of adducts formed between APAP-dosed mice of any genotype. The cumulative data suggest that Cyp1a2 does not play a significant role in APAP hepatotoxicity in these mice.

Cytochrome P4501A2 (CYP1A2) has been identified as a key factor in the metabolic activation of numerous chemical carcinogens, including afiatoxin B1, various heterocyclic and aromatic amines, and certain nitroaromatic compounds. In addition, CYP1A2 contributes to the inactivation of several common drugs and dietary constituents, including acetaminophen and caffeine. Two xenobiotic-responsive-element (XRE)-like sequences and an antioxidant response element (ARE) have been identified in the regulatory region of the CYP1A2 gene; however, the functionality of the ARE remains to be demonstrated. Based on in vivo phenotyping assays, substantial interindividual variability in CYP1A2 activity has been reported. Some population-based studies have reported either bi- or tri-modal distributions in CYP1A2 phenotype, suggesting a genetic basis for the large interindividual differences in CYP1A2 activity. However, despite the polymodal distributions reported for CYP1A2 activity, a distinct functional genetic polymorphism in the gene has not been identified. Potential mechanisms contributing to the large interindividual variability in CYP1A2 activity are discussed. A thorough understanding of the functions and regulation of the CYP1A2 gene may ultimately lead to new methods for preventing or intervening in the development of certain chemically-related human cancers.

Comparison of the effects of biliary lipid excretion produced by infusion of taurochenodeoxycholate and taurocholate showed no significant difference when the bile acids were infused for a relatively short period of time. Cholesterol excretion rates measured during depletion of the bile acid pool were significantly higher than cholesterol excretion rates measured during infusion of bile acids at various rates. These data indicate that there is some mechanism in addition to bile acid excretion that is responsible for biliary excretion of cholesterol when the enterohepatic circulation is intact.The authors wish to acknowledge the technical assistance of Ms. Jean Fahrbach and Ms. Pat Hogan.

Commercially caught crayfish (Pacifasticus leniusculus) were placed in a municipal lake below a combined sewer overflow outfall and a storm drain outfall associated with elevated sediment metal concentrations. Abdominal muscle, viscera, and exoskeleton from each crayfish were analyzed for mercury, cadmium, lead, and copper. Crayfish metal concentrations for each sampling site were evaluated relative to unexposed samples from the commercial catch and samples held in the laboratory. Results indicated that 1) mercury accumulated in muscle tissue, highest cadmium concentrations were in the viscera, and highest lead concentrations were in the exoskeleton, 2) uptake of copper is well-regulated by the organism at non-toxic water concentrations, and 3) viscera concentrations of cadmium, lead, and copper tended to be higher and more variable than in muscle tissue. A significant correlation was found between body weight and muscle mercury concentration. Relative to allowable limits for metals in foods, there was not sufficient accumulation of any metal to indicate that a significant health hazard would result from consumption of these organisms. These data indicate that analysis of trace metals in various body parts ofP. leniusculus may be a useful biological indicator of trace metal pollution of freshwater lakes and streams.

This study investigated the dynamics of 2,4-dichlorophenoxyacetic acid (2,4-D) in Salmonid fish. Similar to mammals and marine fish, trout excrete 2,4-D via the urine, with an elimination half-life of 2.4 hr. Although the amount of 2,4-D found in bile was always less than 1% of the dose, the concentration of 2,4-D in bile was greater than any other tissue or fluid four hr or more after exposure. From 20 to 96 hr following administration, bile was the only tissue or fluid which contained detectable amounts of 2,4-D. The results demonstrate that rainbow trout eliminate 2,4-D very rapidly via urinary excretion. However, detectable levels of 2,4-D may be found in the bile for many hours after it is eliminated from all other tissues.

The combined presence of CYP1A2 and 3A4, both of which oxidize aflatoxin B1(AFB1) to the reactive aflatoxin B1-8,9-epoxide (AFBO) and to hydroxylated inactivation products aflatoxin M1(AFM1) and aflatoxin Q1(AFQ1), substantially complicates the kinetic analysis of AFB1oxidation in human liver microsomes. In the present study, we examine the reaction kinetics of AFB1oxidation in human liver microsomes (HLMs,N= 3) and in human CYP3A4 and CYP1A2 cDNA-expressed lymphoblastoid microsomes for the purpose of identifying the CYP isoform(s) responsible for AFB1oxidation at low substrate concentrations approaching those potentially encountered in the diet. AFBO formation by cDNA-expressed human CYP1A2 followed Michaelis–Menten kinetics (Km= 41 μM, Vmax= 2.63 nmol/min/nmol P450). Furthermore, the portion of AFBO formed in HLMs which was eliminated by furafylline, a specific mechanism-based inhibitor of CYP1A2, also followed Michaelis–Menten kinetics (Km= 32-47 μM, Vmax= 0.36-0.69 nmol/min/nmol P450). The formation of AFBO (activation product) and AFQ1(detoxification product) in cDNA-expressed human CYP3A4 microsomes was sigmoidal and consistent with the kinetics of substrate activation. Accordingly, application of a sigmoid Vmaxmodel equivalent to the Hill equation produced excellent fits to the cDNA-expressed CYP3A4 data and also to the data from HLMs pretreated with furafylline to remove CYP1A2. The Hill model predicted that two substrate binding sites are involved in CYP3A4-mediated AFB1catalysis and that the average affinity of AFB1for the two sites was 140–180 μM. Vmaxvalues for AFQ1formation were 10-fold greater than those for AFBO, and total substrate turnover to both was 67 nmol/min/nmol CYP3A4. Using the derived kinetic parameters for CYP1A2 and 3A4 to model thein vitrorates of AFB activation at low substrate concentrations, it was predicted that CYP1A2 contributes to over 95% of AFB activation in human liver microsomes at 0.1 μMAFB. The important role of CYP1A2 in thein vitroactivation of AFB at low substrate concentrations was supported by DNA binding studies. AFB1–DNA binding in control HLMs (reflecting the contribution of CYP1A2 and CYP3A4) and furafylline-pretreated microsomes (reflecting the contribution of CYP3A4 only) catalyzed the binding of 1.71 and 0.085 pmol equivalents of AFB1to DNA, respectively, indicating that CYP1A2 was responsible for 95% of AFB1–DNA adduct formation at 0.133 μMAFB. These results demonstrate that CYP1A2 dominates the activation of AFB in human liver microsomesin vitroat submicromolar concentrations and support the hypothesis that CYP1A2 is the predominant enzyme responsible for AFBO activation in human liverin vivoat the relatively low dietary concentrations encountered in the human diet, even in high AFB exposure regions of the world. However, because the actual concentrations of AFB in liverin vivofollowing dietary exposures are uncertain, additional studies in exposed human populations are needed. Quantitative data on the relative rates of AFM1and AFQ1excretion (potential biomarkers for CYP1A2 and 3A4 activity, respectively) in humans would be useful to validate the actual contributions of these two enzymes to AFB1oxidationin vivo.

Although epidemiological studies suggest that aflatoxin B1(AFB1) is a human carcinogen, at least in the presence of hepatitis B virus infection, animal studies have demonstrated large differences in species sensitivity to AFB1, and the sensitivity of humans relative to experimental animals remains unclear. The purpose of this study was to determine the profile of AFB1metabolism and the extent of AFB1binding to cell macromolecules in human liver slices under experimental conditions that would allow direct comparison to similar endpoints in the rat, a species sensitive to the carcinogenic actions of AFB1. Liver slices were prepared from three individual human liver samples with a Krumdieck tissue slicer and incubated with 0.5 μM[3H]AFB1for 2 hr. Significant interindividual variations were observed in the rates of oxidative metabolite formation and in specific binding to cell macromolecules. The rates of oxidative metabolism of AFB1to AFQ1, AFP1, and AFM1in the three human liver samples were similar to those previously observed in rat liver slices. AFB1–GSH conjugate formation was not detected in any of the human liver samples, and yet specific binding of AFB1to cell macromolecules was considerably lower in the human liver slices relative to that in rat liver slices. AFB1–DNA binding levels ranged from 3 to 26% of control rat and AFB1–RNA binding levels ranged from 25 to 49% of control rat. The AFB1–protein binding level in the one human sample measured was 20% of that observed for control rat. While these results suggest that humans do not form as much AFBO as the rat, they are also consistent with the hypothesis that humans do not possess GST isozyme(s) with high specific activity toward AFBO. Significant individual differences in AFB1metabolism and binding between humans suggest the presence of genetic and/or environmental factors that may confer large variability in susceptibility to AFB1.

Fibroblasts from patients with the disease 5-oxoprolinuria have reduced glutathione synthetase activity and are thus glutathione (GSH) deficient. In this study, 5-oxoprolinuria fibroblasts (GM3877 cells) contained less GSH than normal diploid fibroblasts as determined by biochemical analysis and by flow cytometry using monochlorobimane. They also contained lower γ-glutamylcysteine synthetase activity than normal cells. However, cocultures of GM3877 cells and normal cells displayed either normal or slightly elevated GSH content, depending upon the assay used. When differentially labeled with fluorescent beads, cocultured, and then isolated by fluorescence-activated cell sorting, both GM3877 cells and normal cells had GSH content similar to that of sorted normal cells cultured alone, whereas sorted GM3877 cells cultured alone showed depressed GSH content. GM3877 cells had detectable levels of γ-glutamylcysteine (γ-GC) when cultured alone, but γ-GC was undetectable in these cells when they were cocultured with normal cells, indicating that it was efficiently metabolized to GSH by the normal cells. These changes in low-molecular-weight thiols were likely to have been mediated by metabolic cooperation across gap junctions because they were dependent upon confluency and because media conditioned by either cell type failed to significantly alter the GSH content of the other cell type. Cocultures exposed to moderate levels of hydrogen peroxide showed less depletion of GSH than GM3877 cells cultured alone, suggesting that the sharing of low-molecular-weight thiols or other reductants via metabolic cooperation can protect cells from oxidative stress. Copyright 1994, 1999 Academic Press

Mice constitutively express glutathioneS-transferase mGSTA3-3 in liver. This isoform possesses uniquely high conjugating activity toward aflatoxin B1-8,9-epoxide (AFBO), thereby protecting mice from aflatoxin B1-induced hepatocarcinogenicity. In contrast, rats constitutively express a closely related GST isoenzyme, rGSTA3-3, with low AFBO activity and, therefore, are sensitive to aflatoxin B1exposure. Although the two GSTs share 86% sequence identity and have similar catalytic activities toward 1-chloro-2,4-dinitrobenzene (CDNB), they have an approximately 1000-fold difference in catalytic activity toward AFBO. To identify amino acids that confer high activity toward AFBO, non-conserved rGSTA3-3 residues were replaced with mGSTA3-3 residues in two regions believed to form the substrate binding site. Twenty-one mutant rGSTA3-3 enzymes were generated by site-directed mutagenesis using combinations of nine different residues. Except for the E208D mutant, single mutations of rGSTA3-3 produced enzymes with no detectable AFBO activity. Generally, AFBO conjugation activity increased in additive fashion as mGSTA3-3 residues were introduced into the rGSTA3-3 enzyme with the six site mutant E104I/H108Y/Y111H/L207F/E208D/V217K displaying the highest AFBO activity (40 nmol/mg/min) of all the mutant enzymes. When this mutant enzyme was further modified by three additional substitutions (D103E/I105M/V106I) AFBO conjugation activity decreased 14-fold to 2.8 nmol/mg/min. Although wild-type mGSTA3-3 AFBO conjugation activity (265 nmol/mg/min) could not be obtained by our rGSTA3-3 mutants, we were able to identify six mGSTA3-3 residues; Ile104, Tyr108, His111, Phe207, Asp208, and Lys217that, when collectively substituted into rGSTA3-3, substantially increased (>200-fold) glutathione conjugation activity toward AFBO.

The binding of two different reaction products (p-nitrobenzyl glutathione and the aflatoxin-glutathione conjugate) to mouse glutathione S-transferase A3-3 (mGSTA3-3) has been measured using equilibrium dialysis and a direct fluorescence quenching technique. As expected, p-nitrobenzyl glutathione was found to bind with a stoichiometry of 2.24 ± 0.17 mol/mol of dimeric enzyme. However, the much larger aflatoxin-glutathione conjugate, 8,9-dihydro-8-(S-glutathionyl)-9-hydroxyl-aflatoxin B1(AFB-GSH), was found to bind with a stoichiometry of 1.12 ± 0.08 mol/mol of dimeric enzyme. p-Nitrobenzyl glutathione bound mGSTA3-3 with a dissociation constant (Kd) of 59 ± 17 µMwhile the aflatoxin-glutathione conjugate bound the enzyme with a Kdof 0.86 ± 0.19 µM. Glutathione competitively inhibited binding of AFB-GSH to mGSTA3-3 with a Kiof 1.5 mM, suggesting that AFB-GSH was binding to the enzyme active site. Although AFB-GSH bound to mGSTA3-3 with a stoichiometry of 1 mol/mol of dimeric enzyme, AFB-GSH completely inhibited activity toward 1-chloro-2,4-dinitrobenzene, indicating that AFB-GSH binding to one active site alters affinity for 1-chloro-2,4-dinitrobenzene in the active site of the other subunit. To our knowledge, this is the first report of a glutathione S-transferase reaction product which binds to the enzyme with a stoichiometry of 1 mol/mol of dimer.

Inhalation exposure to the carcinogen aflatoxin B1(AFB1) in certain occupations is considerable. Because circumstantial epidemiological evidence suggests that AFB1inhalation may cause primary lung cancer, we investigated AFB1activation by human lung microsomes. Microsomes were incubated with [3H]AFB1(124 μm), and activation to the AFB1-8,9-epoxide was measured as the AFB1–glutathione (AFB1–GSH) conjugate by HPLC. The formation of AFB1–GSH was in the range of 0.05–0.073 fmol/mg protein/min. The role of cytochrome P450 (CYP) 3A in this activation was investigated by oxidation of nifedipine (a prototype substrate for CYP 3A), by immunoinhibition, and by immunoblot analysis. Nifedipine oxidation varied from 0.2 to 19.2 pmol/mg protein/min in microsomes from different subjects, but did not correlate with AFB1activation. Anti-human polyclonal CYP 3A4 IgG inhibited AFB1activation. CYP 3A isoforms were immunoestimated to be in the range of 0.01–1.90 pmol/mg protein. Neither CYP 1A2 nor associated activity was detected in the lung microsomes. These data indicate that human lung microsomes activate AFB1to form theexo-AFB1-8,9-epoxide and that CYP(s) of the 3A subfamily may be responsible for this activity. The relatively low amount of AFB1activation in human lung compared to that in human liver can be explained by the scarcity of CYP-containing cells in the lung.In situAFB1activation and resultant carcinogenic risk are distinctly possible in occupational settings where inhalation of AFB1-contaminated dusts occurs.

Plasma levels of renin and pseudorenin were measured at various times in control rats, and after bilateral nephrec-tomy, treatment with propranolol, or the converting enzyme inhibitor (CEI) SQ 20881. After nephrectomy or propranolol, renin levels decreased but pseudorenin levels increased. Following treatment with CEI, renin levels rose in a nonoperated animal and remained very low in a nephrecto-mized animal. Pseudorenin levels did not change appreciably after CEI in either animal.We acknowledge the valuable technical assistance of Mr. John Gillespie and Mrs. Roselle Poisner. We wish to thank E. R. Squibb for the generous supply of SQ 20881.

The liver and the kidney are key toxicity target organs during drug development campaigns, as they typically carry the burden of drug transport and metabolism. Primary hepatocytes and proximal tubule epithelial cells grown in traditional in vitro 2‐D culture systems do not maintain transporter and metabolic functions, thus limiting their utility for nonclinical toxicology investigations. We have developed a renal and hepatic microphysiological system (MPS) platform that uses a commercially available MPS device as the core cell culture platform for our methodologies. We describe protocols for isolating and propagating human proximal epithelial cells and how to seed and culture a renal MPS to recapitulate the human proximal tubule. We present two methods to culture hepatocytes within an MPS and the steps required to connect a renal MPS to a liver MPS. © 2017 by John Wiley & Sons, Inc.

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Glutamate cysteine ligase and gluthione synthase carry out the two-step synthesis of glutathione. The fluorescent thiol-reactive compound monobromobimane is used to derivatize reaction products in an HPLC-based assay with fluorescence detection. The assay described in this unit can be adapted for tissue homogenates or cultured cells.

Results obtained from both epidemiologic studies and experimental animal model systems have shown a wide range of phenotypic variation in the ability of individuals to metabolize drugs and environmental chemicals. Several studies have noted correlations between specific metabolic phenotypes and the incidence of disease, suggesting that certain allelic forms of drug metabolic enzymes can render the individual either more sensitive or resistant to the toxic or therapeutic effects of exogenous drugs and chemicals. While some of this variation can be attributed to different environmental exposures, it has become clear that genetic factors also play an important role in determining the response of the individual organism to exogenous agents. Recent advances in molecular biological techniques have begun to allow scientists to correlate observed phenotypic differences with the actual differences in genetic sequence at the gene level. This has allowed a correlation between gene structure and function, thus providing a mechanistic basis to explain the interaction between genetic background and individual response to environmental exposures. Results presented at this symposium discussed how genetic polymorphisms for both Phase I and Phase II metabolic enzymes in the human population modulate the response to environmental toxicants.

Metabolism of the widely used insecticide methyl parathion by isolated hepatocytes and various subcellular fractions was compared to determine the effects of cellular integrity on the metabolic profile observed. A reverse-phase ion-pair high-performance liquid chromatographic method was developed to separate and quantify methyl parathion and six of its hepatic biotransformation products: methyl paraoxon; desmethyl parathion; desmethyl paraoxon; p-nitrophenol; p-nitrophenyl glucuronide; and p-nitrophenyl sulfate. Most compounds exhibited linear responses and limits of detection below 1 nmol. The chromatographic method was used to determine metabolic profiles of methyl parathion in isolated rat hepatocytes, sonicated hepatocytes, postmitochondrial fraction, microsomes, and cytosol. Isolated hepatocytes produced significantly more desmethyl parathion and p-nitrophenyl sulfate than the subcellular preparations, demonstrating that cellular integrity significantly affects the quantitative metabolic profile observed.

Background There is considerable interindividual variation in the activity of enzymes which metabolize polycyclic aromatic hydrocarbon constituents of tobacco smoke. Low activity of enzymes which detoxify carcinogenic polycyclic aromatic hydrocarbon metabolites may be associated with increased susceptibility to cancers etiologically related to cigarette smoking. Purpose We conducted a population-based, case-control study to determine whether patients with cancers related to smoking had lower activity of detoxifying isoenzymes of glutathione S-transferase (GST) and epoxide hydrolase (EH) than control subjects. Methods Enzyme activities were measured in leukocytes from 113 King County (Washington) residents diagnosed during 1987 with one of three smoking-related cancers (lung, oropharynx/oral cavity, or bladder), 50 King County residents with cancers believed unrelated to smoking (prostate cancer or non-Hodgkins lymphoma), and 120 persons selected at random from the King County population. Enzyme activity measurements were made for leukocyte cytosolic GST toward trans-stilbene oxide (TSO), 1-chloro-2,4-dinitrobenzene, and benzo[a]pyrene-4,5-oxide (BaPO), and for microsomal EH toward BaPO. Results Overall, the distribution of activity levels of GST toward TSO and BaPO did not differ in case patients with smoking-related cancer compared with control subjects. The activities of GST toward 1-chloro-2,4-dinitro-benzene and of EH toward BaPO were somewhat lower on average in case patients with smoking-related cancers than in control subjects, but these differences were well within the limits of chance. Among the heaviest smokers, there were proportionately fewer patients with smoking-related cancers than control subjects with intermediate or high GST activity toward TSO (odds ratio = 0.6), but this difference was also plausibly due to chance (95% confidence interval = 0.3–1.1). Conclusions While the findings of this study are compatible with a moderate protective effect of high or intermediate enzyme activity among persons heavily exposed to tobacco, as suggested by an earlier report, the data are by no means conclusive. [J Natl Cancer Inst 84:414–422, 1992]