Induction of rat liver microsomal epoxide hydrolase by thiazole and pyrazine: hydrolysis of 2-cyanoethylene oxide

Liver microsomal epoxide hydrolase (mEH) is active in the detoxificationof epoxide-containing carcinogens. The effects of thiazole andpyrazine, constituents of tobacco and tobacco smoke as wellas of a variety of foods, on the expression and regulation ofmEH were examined in rats (200 mg/kg body wt/day, i.p., 1/emdash3 days). Immunoblot analyses using rabbit anti-rat mEH antibodyrevealed a significant increase in mEH levels in hepatic microsomesisolated from either thiazole- or pyrazine-treated animals.Another protein (43 kd) cross-reacting with polyclonal mEH antibodywas found to be increased concomitantly following pyrazine treatment.Northern and slot blot analyses showed substantial increasesin mEH mRNA following either thiazole or pyrazine treatment.The level of mEH mRNA increased 17-fold at 24 h following thiazoletreatment, relative to control. Approximately 20- and 16-foldincreases in mEH mRNA were also observed at 48 and 72 h respectivelyfollowing treatment with pyrazine. The level of polymerase chainreaction (PCR)-amplified mEH DNA derived from poly(A)+ RNA wasclearly elevated following either thiazole or pyrazine treatmentrelative to that from untreated animals. Both sense and antisensestrands of PCR-amplified mEH DNA were cloned into an M13mpl9phage vector in order to examine the nucleotide sequences ofPCR-amplified mEH DNA derived from the poly(A)+ RNA isolatedfrom thiazole- or pyrazine-treated animals. Sequence analysesrevealed that the sequence of PCR-amplified DNA from the inducedmRNA was identical to that published for mEH cDNA. Epoxide hydrolaseactivity toward the hydrolysis of 2-cyanoethylene oxide (CEO),the epoxide metabolite of the rat carcinogen acrylonitrile,was not significant in hepatic microsomes from untreated rats,but was substantially induced by treatment with thiazole orpyrazine. Microsomal hydrolysis activity was heat-sensitiveand potently inhibited by l, l, l-trichloropropene-2, 3-oxide,indicating that mEH was the catalyst. The V_max for the hydrolysisof CEO by hepatic microsomes from thiazole-treated rats (13.4nmol/min/mg protein) was 1.5-fold greater than that with microsomesfrom pyrazine-treated rats, whereas similar K_m values ( 1 mM)were observed for both microsomal preparations. These kineticdata correlate well with the increases in mEH mRNA observedafter administration of thiazole or pyrazine to rats. Theseresults provide evidence that administration of thiazole orpyrazine induces mEH with a large increase in mEH mRNA, andthat the induced mEH catalyzes the hydrolysis of CEO.     

Comparison of the biotransformation of 1,3-butadiene and its metabolite, butadiene monoepoxide, by hepatic and pulmonary tissues from humans, rats and mice.

1,3-Butadiene (BD), a widely used monomer in the production of synthetic rubber and other resins, is one of the 189 hazardous air pollutants identified in the 1990 Clean Air Act Amendments. BD induces tumors at multiple organ sites in B6C3F1 mice and Sprague-Dawley rats; mice are much more susceptible to the carcinogenic action of BD than are rats. Previous in vivo studies have indicated higher circulating blood levels of butadiene monoepoxide (BMO), a potential carcinogenic metabolite of BD, in mice compared to rats, suggesting that species differences in the metabolism of BD may be responsible for the observed differences in carcinogenic susceptibility. The metabolic fate of BD in humans is unknown. The objective of these studies was to quantitate in vitro species differences in the oxidation of BD and BMO by cytochrome P450-dependent monooxygenases and the inactivation of BMO by epoxide hydrolases and glutathione S-transferases using microsomal and cytosolic preparations of livers and lungs obtained from Sprague-Dawley rats, B6C3F1 mice and humans. Maximum rates for BD oxidation (Vmax) were highest for mouse liver microsomes (2.6 nmol/mg protein/min) compared to humans (1.2) and rats (0.6). The Vmax for BD oxidation by mouse lung microsomes was similar to that of mouse liver but greater than 10-fold higher than the Vmax for the reaction in human or rat lung microsomes. Correlation analysis revealed that P450 2E1 is the major P450 enzyme responsible for oxidation of BD to BMO. Only mouse liver microsomes displayed quantifiable rates for metabolism of BMO to butadiene diepoxide (Vmax = 0.2 nmol/mg protein/min), a known rodent carcinogen. Human liver microsomes displayed the highest rate of BMO hydrolysis by epoxide hydrolases. The Vmax in human liver microsomes ranged from 9 to 58 nmol/mg protein/min and was at least 2-fold higher than the Vmax observed in mouse and rat liver microsomes. The Vmax for glutathione S-transferase-catalyzed conjugation of BMO with glutathione was highest for mouse liver cytosol (500 nmol/mg protein/min) compared to human (45) or rat (241) liver cytosol. In general, the KMs for the detoxication reactions were 1000-fold higher than the KMs for the oxidation reaction. Because of the low solubility of the BD and the relatively high KM for oxidation, it is likely that the Vmax/KM ratio will be important for BD and BMO metabolism in vivo. In vivo clearance constants were calculated from in vitro data for BD oxidation and BMO oxidation, hydrolysis and GSH conjugation.(ABSTRACT TRUNCATED AT 400 WORDS)     

In vitro binding of aflatoxin B1 and 2-acetylaminofluorene to rat, mouse and human hepatocyte DNA: the relationship of DNA binding to carcinogenicity

DNA binding levels were determined and compared in culturedhepatocytes from male and female rats as well as other animalspecies following exposure to aflatoxin B₁ (AFB₁) or 2-acetylaminofluorene(2-AAF). When human, rat (both male and female) and mouse hepatocytesin primary culture were exposed to 2.0 ? 10^–7 M [³H]AFB₁(sp. act. 2.63 µCi/nmol) for 24 h, male rat hepatocyteshad the highest degree of [³H]AFB₁-DNA binding (203 pmol/mgDNA) and human hepatocytes contained the next highest bindinglevel (42 pmol/mg DNA). Hepatocytes from female rats contained38 pmol/mg DNA while cultured mouse hepatocytes contained only1.4 pmol/mg DNA. When the same dose of [³H]AFB₁ was administeredto the cultured male rat hepatocytes at 24 h, 48 h, 72 h and1 week after seeding, and incubated for 24 h, the DNA bindinglevels were 189, 175, 76, 75 pmol/mg DNA respectively. In parallelexperiments to the cultured male rat hepatocytes above, theAFB₁-DNA binding levels in the cultured female hepatocytes were42, 41, 37 and 34 pmol/mg DNA respectively. Human, male andfemale rat hepatocytes in primary culture were exposed to 5.2? 10^–5 M 2-acetyl-amino [9–¹⁴C]fluorene (sp. act.0.0094 µCi/nmol) for 24 h. It was determined that malerat hepatocytes contained the highest amount of radioactivelylabeled 2-AAF bound to their DNA (1.57 nmol/mg DNA), femalerat hepatocytes contained 0.62 nmol/mg DNA and human hepatocytescontained 0.29 nmol/mg DNA. Results from our in vitro hepatocyteculture system correlate well with in vivo animal studies dealingwith species and sex differences in DNA binding and carcinogenicsusceptability. This indicates that hepatocytes in vitro maintainmany of the biological properties necessary for carcinogen responsesimilar to liver cells in vivo. In addition, comparison of genotoxiceffect in cultured hepatocytes from animals as well as humansmay be useful in evaluating carcinogenic potential of xenobioticsin human liver.     

Enzymatic reduction of chromium(VI) by human hepatic microsomes

The reduction of chromium(VI) by human hepatic microsomes wasinvestigated. The reduction rates were proportional to the amountof microsomes added and reduction was mediated by an NADPH-dependentenzymatic system which exhibited a K_m for chromate of 1.04±0.18µM and a V_max of 5.03±0.49 nmol/min/mg protein.Relative to incubation under 0% O₂, 21% O₂ inhibited microsomalCr(VI) reduction in three individuals by 53, 36 and 37%. Cr(VI)reduction was not inhibited by metyrapone, carbon monoxide,aminopyrine, piperonyl butoxide or chloroform, suggesting thatcytochrome P450s did not play a major role. Thallium trichloride(0.13 and 0.26 mM), a known flavoprotein inhibitor, caused acomplete inhibition of both Cr(VI) reduction and NADPH:cytochromeP450 (c) reductase activity. A partial inhibition of Cr(VI)reduction was seen in the presence of n-octylamine, which maysuggest a possible role for flavin-containing monooxygenase(FMO). Overall, human microsomal Cr(VI) reduction is very differentfrom the P450-mediated microsomal reduction observed in rodents.Specifically, the human system is much less oxygen-sensitive,has a much greater affinity for chromate and is apparently mediatedby flavoproteins.     

Human liver glucuronidation of benzidine

Although glucuronidation is considered an important pathwayin aromatic amine-induced bladder cancer, benzidine glucuronidationhas not been assessed in humans. Glucuronidation of benzidinewas assessed with human liver microsomes and slices. Emulgen911-treated microsomes exhibited a K_m for benzidine of 0.8±0.06mM and a V_max of 4.2±0.7 nmol/mg protein/min. A varietyof agents were tested for their ability to inhibit benzidineN-glucuronide formation. At 0.25 mM, estriol, 17-epiestriol,bilirubin, hyodeoxycholic acid and cyproheptadine were goodinhibitors (<50% of control). Dose-dependent inhibition studieswith estriol, testosterone and 4-aminobiphenyl demonstratedthat each agent reached a plateau as its concentration was increased.When these agents were combined at maximal inhibitory concentrations,additive inhibition was observed. These results suggest thatmore than one UDP-glucuronosyltransferase metabolizes benzidine.The cDNA clones pUDPGT_h-1 and -2 encode transferases which metabolizehyodeoxycholic acid and estrogen derivatives, but neither transferasecatalyzed benzidine glucuronidation. Slices were used to assessmetabolism by intact tissue and converted [³H]benzidine (0.09mM) to N-acetyl-benzidine. N-Glucuronides of both benzidineand N-acetylbenzidine were observed and represented 14–37%of the total recovered radioactivity. The amount of N-acetylbenzidineN'-glucuronide observed was proportional to the amount of N-acetylbenzidineproduced. Thus, N-glucuronidation appears to represent a majorpathway for metabolism of benzidine in humans. The extent ofN-acetylation affects the proportion of benzidine and N-acetylbenzidineglucuronidated by human liver slices.     

Epoxide reductase activity of mammalian liver cytosols and aldehyde oxidase

The present study provides the first evidence that a mammalianliver cytosolic enzyme, aldehyde oxidase, has an ability toreduce arene oxides to the parent hydrocarbons under anaerobicconditions. The comparative ability of rabbit liver preparationsto reduce arene oxides was examined using naphthalene 1,2-oxideand benzo[a]pyrene 4,5-oxide as substrates. The liver cytosolwith an electron donor of aldehyde oxidase exhibited much higherepoxide reductase activity compared with the liver microsomeswith NADPH and FAD. The cytosolic activity was sensitive toinhibitors of aldehyde oxidase. Purified rabbit liver aldehydeoxidase also exhibited a significant epoxide reductase activityin the presence of its electron donor. Apparent K_m. and V_maxvalues of the enzyme were 426 µM and 323 nmol/min/mg proteinfor naphthalene 1,2-oxide and 255 µM and 100 nmol/min/mgprotein for benzo[a]pyrene 4,5-oxide respectively. However,no epoxide reduction by the enzyme or by the liver cytosol wasdetected in olefin epoxides such as styrene oxide and trans-stilbeneoxide. Similar results were obtained with rat liver preparations.However, the epoxide reductase activity of cytosol and aldehydeoxidase from rat liver was considerably lower than that of therabbit liver preparations. In hamsters, mice and guinea-pigs,liver cytosols with an electron donor of aldehyde oxidase aswell as liver microsomes with NADPH exhibited a significantepoxide reductase activity toward naphthalene 1,2-oxide. However,no epoxide reduction was observed with dog liver cytosol. Administrationof sodium tungstate to rats depleted liver cytosolic reductaseactivity and sodium molybdate treatment resulted in partialrestoration of the activity, supporting the view that the epoxidereductase activity observed in the liver cytosol mainly originatesfrom aldehyde oxidase.     

Time-related binding of the hepatocarcinogen carbon tetrachloride to hepatic chromatin proteins in vitro

The in vitro covalent binding of ¹⁴C-labelled carbon tetrachloride[¹⁴C]CCl₄ to histones and non-histone chromosomal proteins (NHCP)under microsome-mediated aerobic conditions was determined.Whole chromatin was prepared from purified nuclei isolated fromlivers of B6C3F1 hybrid mice and incubated with 2.5, 5.0 and10.0 µmol [¹⁴C]CCl₄, in the presence of microsomes isolatedfrom the same tissue, at 4 mg protein, and an NADPH-regeneratingsystem at 37°C for varying incubation times. Binding of[¹⁴C]CCl₄ to histones and NHCP was also determined in the presenceof 5 mM L-cysteine. The results show that the activated intermediateof CCl₄ bound more to histones than to NHCP in a dose- and time-dependentmanner, and that 5 mM L-cysteine inhibited the binding of theactivated intermediate of CCl₄ to histones by 59%, without affectingthe binding to NHCP. These data suggest different extents ofalkylation or acylation between histones and NHCP by metabolicallyactivated CCl₄ under aerobic in vitro conditions, and differentialinhibition of CCl₄-alkylation-acylation by cysteine. This suggestiondoes not exclude other possible mechanisms of action.     

NADPH-dependent oxidation of benzidine by rat liver

This study used liver microsomes from control and ß-naphthoflavone-treatedrats to evaluate NADPH-dependent oxidation of benzidine. Withmicrosomes from ß-naphtho-flavone-treated rats, therates of formation of aqueous soluble metabolite (HPLC analysis)and protein and DNA binding were 835 ±81, 14.5 ±1.8 and 0.71 ± 0.14 pmol/ mg/min respectively. ß-Naphthoflavonetreatment elicited 12.3-, 1.8- and 14.2-fold increases in benzidinemetabolism compared with controls as judged by HPLC and proteinand DNA binding respectively. For microsomes from treated animals,K_m and V_max values were 47 ± 6 µM and 1.13 ±0.16 nmol/mg protein/min respectively. All of the metabolicparameters were inhibited to varying degrees by gluta-thione(1 or 10 mM), N-acetylmethionine (10 mM) and ascorbic acid (10mM). Following glutathione addition, at least two new metabolitepeaks were observed, representing     

Anaerobic metabolism and nuclear binding of the carcinogen 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT)

The anaerobic reductive metabolism of the urinary tract carcinogen2-amino-4-(5-nitro-2-furyl)-[2-¹⁴C]-thiazole ([¹⁴C]ANFT) wasexamined in vitro using rabbit liver and kidney microsomes.The intermediate(s) produced during the reduction binds to tRNA,DNA, and protein. ANFT reduction was inhibited by oxygen, requiredNADPH and was not inhibited by SKF-525A or allopurinol. No bindingto tRNA or DNA was observed if the nudeic acids were added atthe end of the incubation. The covalent binding of an ANFT metabolite(s)to nucleic acids and protein was inhibited by the antioxidantsvitamin E and butylated hydroxytoluene. The stoichiometry ofmicrosomal reduction shows 3 mol of NADPH were used/mol of ANFTreduced. In inner medullary microsomes, the apparent K_m andV_max were 0.05 mM and 0.92 nmol/mg/min, respectively. Two metabolitesfrom the anaerobic incubation of ANFT were isolated. The metaboliteswere tentatively identified as 1-(2-amino-4-thiazolyl)-3-cyano-1-propanoneand 2-amino-4-(5-hydroxylamino-2-furyl)thiazole.     

Modulation of microsome-mediated aflatoxin B1 binding to exogenous and endogenous DNA by cytosolic glutathione S-transferases in rat and hamster livers

Microsome mediated aflatoxin B₁ (AFB₁) binding to exogenousand endogenous DNA and its modulation by cytosolic glutathione(GSH) S-transferases have been examined in rat and hamster livers.Kinetic studies over a wide range of cytosol concentrationsindicate that cytosol from the hamster is several-fold moreeffective than that from the rat in inhibiting AFB₁ bindingto exogenous calf thymus DNA mediated by microsomes from eitherspecies. Low concentrations of GSH (0.1–0.2 mM) are requiredfor 50% inhibition of AFB₁—DNA binding by cytosol. Withexogenous DNA, combined microsome-cytosol fractions from thehamster give more AFB₁—DNA binding than those from therat. However, with nuclei as a source of endogenous DNA, AFB₁—DNAbinding is less with combined microsome-cytosol fractions fromthe hamster than those from the rat. Cytosolic inhibition ofAFB₁—DNA binding is almost completely reversed in thepresence of 1 mM levels of either trichloropropene oxide orstyrene oxide. Quantitation of AFB₁—DNA binding and AFB₁-GSH conjugation indicate an inverse relationship between thesetwo processes. Cytosol from the rat has less capacity than thatfrom the hamster to form an AFB₁—GSH conjugate. HepaticGSH levels are about equal (6–7 mM) in both species. I.p.administration of [¹⁴C]AFB₁ 2 h before sacrifice gives moreAFB₁ binding to hepatic nuclear DNA in rats than in hamsters.However, depletion of hepatic GSH levels by 80% by i.p. administrationof diethylmaleate (600 mg/kg) increases AFB₁—DNA binding2- to 3-fold in both species. The role of cytosolic GSH S-transferasesin modulating hepatic AFB₁—DNA binding in rats and hamstersis discussed.     

Biotransformation of aflatoxin B1 in human lung

In addition to being a potent hepatocarcinogen, aflatoxin B₁(AFB₁) is a pulmonary carcinogen in experimental animals, andepidemiological studies have shown an association between AFB₁exposure and lung cancer in humans. This study investigatedAFB₁ bioactivation and detoxification in human lung tissue obtainedfrom patients under-going clinically indicated lobectomy. [³H]AFB₁was bioactivated to a DNA binding metabolite by human wholelung cytosols in a time-, protein concentration-, and AFB₁ concentration-dependentmanner. Cytosolic activation of [³H]AFB₁ correlated with lipoxygenase(LOX) activity and was inhibited by the LOX inhibitor nordihydroguaiareticacid (NDGA; 100 µM), indicating that LOXs were largelyresponsible for the observed cytosolic activation of AFB₁. Inwhole lung microsomes, low levels of indomethacin inhibitableprostaglandin H synthase (PHS)-mediated [³H]AFB₁-DNA bindingand cytochrome P-450 (P450)-mediated [³H]AFB₁-DNA binding wereobserved. Cytosolic glutathione S-transferase (GST)-catalyzeddetoxification of AFB₁–8,9-epoxide, produced by rabbitliver microsomes, was minimal at 1 and 10 µM [³H]AFB₁.With 100 µM [³H]AFB₁, [³H]AFB₁–8, 9-epoxide conjugationwith reduced glutathione was 0.34 ± 0.26 pmol/mg/h (n= 10). In intact, isolated human lung cells, [³H]AFB₁ bindingto cellular DNA was higher in cell fractions enriched in macrophagesthan in either type II cell-enriched fractions or fractionscontaining unseparated cell types. Indomethacin produced a 63–100%decrease in [³H]AFB₁-DNA binding in macrophages from five ofseven patients, while NDGA inhibited [³H]AFB₁ -DNA adduct formationby 19, 40 and 56% in macrophages from three of seven patients.In alveolar type O cells, NDGA decreased [³H]AFB₁-DNA bindingby 30–100% in cells from three patients and indomethacinhad little effect. SKF525A, an isozyme non-selective P450 inhibitor,enhanced [³H]AFB₁ binding to cellular DNA in unseparated cells,macrophages, and type II cells, suggesting that P450-mediatedbioactivation of AFB₁ is not a major pathway by which AFB₁–8,9-epoxideis formed in human lung cells. Overall, these studies suggestthat P450 has a minor role in the bioactivation of AFB₁ in humanlung. Rather, LOXs and PHS appear to be important bioactivationenzymes. Co-oxidative bioactivation of AFB₁, in combinationwith the low conjugating activity displayed by human lung cytosolicGSTs, likely contributes to human pulmonary susceptibility toAFB₁.     

In vitro conjugation of benzene metabolites by human liver: potential influence of interindividual variability on benzene toxicity

In addition to industrial sources, benzene is present in theenvironment as a component of cigarette smoke and automobileemissions. Toxicity of benzene most likely results from oxidativemetabolism of benzene to reactive products. However, susceptibilityto these toxic effects may be related to a balance between activation(phase I) and detoxication (phase II) reactions. In the presentstudy, we have estimated kinetic parameters of the two majordetoxication reactions for benzene metabolites—phenolsulfation and hydroquinone glucuronidation—in liver subcellularfractions from 10 humans, and single samples from mice and rats.The extent of oxidative metabolism of benzene by these liversamples has been reported previously. Here, initial rates ofphenol sulfation varied 3-fold (range 0.309–0.919 nmol/mgprotein/min) among human samples. Measured rates were fasterin rats (1.195 nmol/mg protein/min) than in mice (0.458 nmol/mgprotein/min). Initial rates of hydroquinone glucuronidationby human samples also varied 3-fold (range 0.101–0.281nmol/mg protein/min). Hydroquinone glucuronidation was morerapid by mouse microsomes (0.218 nmol/ mg protein/min) thanby rat microsomes (0.077 nmol/mg protein/min). To integrateinterindividual differences in various enzyme activities, aphysiological compartmental model was developed that incorporatesrates of both conjugation reactions and oxidation reactions.Model equations were solved for steady-state concentrationsof phenol and hydroquinone attained in human, mouse and ratblood during continuous exposure to benzene (0.01 µM inblood). Among the 10 human subjects, steady-state concentrationsof phenol varied 6-fold (range 0.38–2.17 nM) and steady-stateconcentrations of hydroquinone varied 5-fold (range 6.66–31.44nM). Predicted steady-state concentrations of phenol were higherin mice compared with rats (2.28 and 0.83 nM respectively).Likewise, higher steady-state concentrations of hydroquinonewere predicted in mice than in rats (42.44 and 17.99 nM respectively).Predicted steady-state concentrations of phenol and hydroquinonein mice were higher than predictions for the 10 human subjects,whereas predicted concentrations for rats fell among the humanvalues. As such, our results underscore the importance of consideringthe balance between activation and detoxication reactions inthe elimination of toxicants. Model simulations suggest thatboth phase I and phase II pathways influence the relative riskfrom exposure to benzene.     

Labeled 1,N6-ethenoadenosine and 3,N4-ethenocytidine in hepatic RNA of mice given [ethyl-1,2-3H or ethyl-1-14C] ethyl carbamate (urethan)

Injection of a single dose of [ethyl-1,2-³H] or [ethy1-1-¹⁴C]-ethylcarbamate into 12-day old male [C57BL/6 x C3H/He]F₁ mice orof [ethyl-1,2-³H]ethyl carbamate into adult male A/Jax miceresulted in the formation of labeled 1,N6-ethenoadenosine and3,N⁴-ethenocytidine adducts in the hepatic RNA. These adductswere characterized by comigration on h.p.l.c. of ³H or ¹⁴C inenzymatic hydrolysates of the RNA with synthetic standards.Both the ethenoadenosine and ethenocytidine were further characterizedby their conversion to acetylated products that comigrated withacetylated synthetic standards. The ethenoadenosine was alsoconverted by anhydrous trifluoroacetic acid to a product thatcomigrated with synthetic 1,N⁶-ethenoadenine. The levels ofadducts in the hepatic RNA 12 h after a single injection of0.5–0.6 mg of ethyl carbamate/g body weight were 6–10and 2–3 pmol/mg RNA of ethenoadenosine and ethenocytidine,respectively. No labeled ethenoadenosine or ethenocytidine couldbe detected in the hepatic RNA of mice given [1-¹⁴C]ethanol,an enzymatic hydrolysis product of ethyl carbamate. These dataindicate that ethyl carbamate may be metabolically activatedby dehydrogenation to vinyl carbamate and subsequent epoxidationof the latter compound as previously proposed. Vinyl carbamateepoxide may form etheno derivatives in a manner analogous tothat demonstrated for chloroethylene oxide, an electrophilicmetabolite of vinyl chloride. Vinyl carbamate has been shownto have the same spectrum of tumor induction as ethyl carbamatebut to be much more active than the latter carcinogen.     

Effect of diet and route of administration on the DNA binding of aflatoxin B1 in the rat

The effects of dietary Brussels sprouts and indole-3-carbinol(I3C) on xenobiotic-metabolizing enzyme activities and hepaticaflatoxin B₁ (AFB₁)-DNA binding were detennined in rats. Animalswere dosed intraperitoneally (i.p.) or intragastrically (i.g.)with [³H]AFB₁ and killed 2 (i.p.) or 3 (i.g.) h later. Brusselssprouts caused a significant (P < 0.01) 50–60% decreasein hepatic AFB₁ binding, and increased hepatic and intestinalglutathione S-transferase (GST) activities. Hepatic mono-oxygenase(AHH and ECD) activities were not altered in sprouts-fed rats,but >2-fold increases in intestinal AHH and ECD activitieswere found. Although I3C increased intestinal AHH and ECD activitiessimilarly to Brussels sprouts, I3C did not significantly decreaseAFB₁ binding, nor did it increase hepatic or intestinal GSTactivity. Route of administration did not alter the percentageinhibition of binding in comparison to control rats in eithertreatment group, suggesting that the small intestine may notplay a significant role in the metabolism of AFB₁. In a secondexperiment, rats were dosed either i.p. or i.g. with [³H]AEB₁and killed 2, 6, 12, 24 or 48 h later. Hepatic AFB₁-DNA bindingand tissue radioactivity levels were determined. Brussels sproutsonce again significantly (P<0.001) decreased hepatic AFB₁-DNAbinding. Route of administration of the carcinogen did not affectDNA binding over time in sprouts-fed animals, confirming ourprevious results.     

A comparative study of the biochemical properties of human and mouse recombinant O6-methylguanine-DNA methyltransferases

The O⁶-methylguanine-DNA methyltransferase (MGMT) repairs mutagenicand carcinogenic O⁶-alkylguanine in DNA by accepting stoichiometricallythe alkyl group from the base. Although the mouse MGMT is largerthan the human protein because of an additional tetrapeptidesequence, these proteins are 70% homologous. Recombinant MGMTsof the human, the mouse and a mouse mutant with the tetrapeptidedeleted were purified to homogeneity from Escherichia coli.The N-terminal amino acid sequences of these proteins are identicalto those predicted from the nucleotide sequences, and theirmolecular masses deter mined by SDS-PAGE agreed with the predictedvalues. However, the observed isoelectric points of 9.3, 9.2and 9.3, for the human, mouse and mutant mouse proteins respectivelywere significantly different from the values, 8.09, 7.47 and7.49 calculated from the amino acid composition. The extinctioncoefficients E_1%^{280 nm} of human, mouse and mutant mouse proteinwere calculated from amino acid composition to be 18.2, 11.1and 11.3 respectively. These values agree fairly well with calculatedvalues. Human and wild-type mouse MGMTs react with the alkylatedbase in a synthetic DNA substrate poly(dC, dG, m⁶dG) with comparablesecond-order rate constants of 2.2x10⁸ and 3.7x10⁸ 1/M/min at37°C respectively and were inactivated by O⁶-benzylguanineat similar rates. The initial reaction rate (K_in) and rate ofinactivation (k_inact) constants for reaction with the base werecalculated to be 1.8x10^–4 M and 1.4x10^–3/s for thehuman protein, 2.3x10^–4 M and 1.1x10^–3/s for thewild-type mouse protein, and 2.1x10^–4 and 1.4x10^–3/sfor the mutant mouse protein respectively. The MGMTs were inactivatedto the extent of 55—65% after heating at 50°C in 20mMTris-HCI, pH 8.0, 1 mM EDTA, 1 mM DTT and 10% glycerol. However,in the presence of DNA (200 µg/ml), only 25—35%of the protein was inactivated. Both DNA and RNA inhibited allthree enzymes in a concentration-dependent fashion, althoughDNA was a better inhibitor than RNA. High salt (0.2 M NaCl)inhibited human MGMT by 80%, while the wild-type and the mutantmouse MGMTs were inhibited by 55%. The human protein had higheraffinity for binding to duplex DNAs than the mouse proteins.     

Species comparison of acrylonitrile epoxidation by microsomes from mice, rats and humans: relationship to epoxide concentrations in mouse and rat blood

Acrylonitrile (ACN) has been shown to cause tumors of the brain, stomach and Zymbal's gland in rats in several bioassays, but it has not been tested in other species. The carcinogenic risk of humans exposed to ACN is unclear. ACN is metabolized in the liver to 2-cyanoethylene oxide (CEO), which is believed to be the proximate or ultimate carcinogenic species. Therefore, the kinetics of CEO formation were studied with liver and lung microsomes from mice and humans using a GC-MS assay for CEO, and the data were compared with previously obtained kinetic parameters for rat microsomal enzymes. The rate of CEO formation by human liver microsomes was comparable to that of rat liver microsomes, but less than that of mouse liver microsomes. Liver microsomes produced more CEO than lung microsomes with all three species. CEO formation by microsomes from mice was approximately 4 times greater than that by microsomes from rats or humans, suggesting that mice would have higher CEO concentrations in blood than rats after ACN exposure. However, after oral administration of ACN, the concentration of CEO in mouse blood was one-third that in rat blood at all doses and time points examined. These results show that CEO circulates via the blood, providing exposure to distant sites. The blood concentrations of CEO do not appear to correlate with rates of microsomal CEO formation. This suggests that species differences in the detoxication of CEO may play an important role in determining circulating CEO concentrations and distant organ exposure.     

Characterization of covalent binding of N'-nitrosonornicotine in rat liver microsomes

The metabolism of the carcinogenic nitrosamine, N'-nitrosonornicotine(NNN), to reactive intermediates which bind covalently was assessedusing male Sprague-Dawley rat liver microsomes. The NADPH-dependentcovalent binding of [¹⁴C]NNN was linear with time up to 90 minand protein concentration up to 3.0 mg/ml. The apparent K_m andV_max of the binding were determined from the initial velocitiesand found to be 0.91 mM and 4.7 pmol/min/mg protein, respectively.Although NNN is not a hepatocarcinogen, this amount of NADPH-dependentcovalent binding is 7-fold greater than that reported for dimethylnitrosamine,a potent hepatocarcinogen. Extensive covalent binding of [¹⁴C]NNNto liver and muscle microsomal protein was also present in theabsence of an NADPH-generating system and in the presence of50% methanol, indicating a non-enzymatically mediated reaction.Addition of the nucleophiles glutathione, cysteine and N-acetylcysteinesignificantly decreased (p <0.01) the non-NADPH-dependentbinding, but did not affect NADPH-dependent binding. In vitroaddition of the cytochrome P-450 inhibitors metyrapone, piperonylbutoxide and SKF-525A significantly decreased (p <0.05) NADPH-dependentbinding of [¹⁴C]NNN by 27–40%. NADH did not replace NADPHin supporting covalent binding. Replacement of an air atmospherewith nitrogen or CO: O₂(8: 2) significantly decreased (p <0.05)NADPH-dependent binding of [¹⁴C]NNN by 40 and 27%, respectively.Aroclor 1254 pre-treatment of the rats did not enhance the NADPH-dependentbinding of [¹⁴C]NNN. These data indicate that cytochrome P-450is at least in part responsible for the metabolic activationof the carcinogen NNN but also suggest additional mechanismsof activation.     

Limited sampling models for topotecan pharmacokinetics

BACKGROUND: Limited sampling models for the estimation of the topotecanArea Under the concentration versus time Curve (AUC) and itslactone ring opened form (AUC Tm), from one or more plasma concentrationdeterminations, are desired for further population-kinetic studies. PATIENTS AND METHODS: The models were developed and validated using 34 pharmacokineticcurves in 19 patients who participated in a phase I study. RESULTS: A single point model was selected as optimal: AUC (µmol/L. min) = 499(min) . C_2h(µmol/L) + 0.85(m²/mg . µmol/L. min) . dose(mg/m²), and for topotecan-metabolite (Tm), AUCTm(µmol/L.min) = 55.1 (min) . CTm_2h (µmol/L) / -0.011(m²/mg. µmol/L . min) . dose (mg/m²), where C_2h is the plasmaconcentration (µmol/L) of topotecan at 2 h after the endof a 30-min infusion, and CTm_2h the concentration of the openedform at the same time point. The models are valid for dosagesranging from 0.5 to 1.5 mg/ m²/day and proved to be unbiased(MPE% = –1.8% and –9.3%, respectively) and precise(RMSE% – 17.9% and 22.7%, respectively). From the predictedAUCs, the clearance (Cl = dose (µmol)/AUC(µmol/L. min)) could also reliably be predicted, as well as the totalAUC (AUC + AUC Tm) (RMSE% = 17.1% and MPE% = –0.02%).Half-life values could not be predicted with acceptable precisionand accuracy. CONCLUSION: The limited sampling models presented may be useful for futurestudies focused on pharmacokinetic/ pharmacodynamic relationshipsof topotecan in large populations. AUC, limited sampling model, pharmacokinetics, topotecan     

N-Sulfooxy-2-aminofluorene is the major ultimate electrophilic and carcinogenic metabolite of N-hydroxy-2-acetylaminofluorene in the livers of infant male C57BL/6J x C3H/HeJ F1 (B6C3F1) mice

The hepatic DNA of 12-day-old male B6C3F₁ (C57BL/6J x C3H/HeJ)mice given an i.p. dose of 0.06 or 0.11 µmol/g body weightof N-hydroxy-[³H]-2-acetylaminofluorene (N-hydroxy-AAF) containedat 9 h 3 or 6 pmol of N-(deoxyguanosin-8-yl)-2-aminofluoreneadducts per mg. Together the level of the two acetylated adductsN-(deoxyguanosin-8-yl)-2-acetyl-aminofluorene and 3-(deoxyguanosin-N2-yl)-2-acetylaminofluorenewas 10% of this amount. The same doses of unlabeled carcinogeninduced by 10 months a 100% incidence of hepatomas with averagesof 10 and 15 hepatomas per mouse, respectively. Injection of0.04 µmol/g body weight of pentachlorophenol (PCP) 45min before the dose of N-hydroxy-AAF decreased the number ofadducts in the DNA by 90% and the average number of hepatomasper liver by 80–90%. As compared to their normal malelittermates, male brachymorphic B6C3F₂ mice, which are deficientin hepatic 3'-phosphoadenosine-5'-phosphosulfate (PAPS), treatedwith N-hydroxy-AAF formed only 25% as many hepatic DNA adductsand developed only 10% as many hepatomas. Hepatic cytosols from12-day-old B6C3F₁ mice contained PAPS-dependent sulfotransferaseactivity for N-hydroxy-2-aminofluorene (N-hydroxy-AF), a previouslyunrecognized activity, as well as sulfotransferase activityfor N-hydroxy-AAF; both activities were inhibited 60% by 1 µMand 80% by 10 µM PCP. Cytosolic acetyl coenzyme A-dependentacetyltransferase activity for N-hydroxy-AF, cytosolic N,O-acyltransferaseactivity for N-hydroxy-AAF, and microsomal deacetylase for N-hydroxy-AAFwere not significantly inhibited by PCP under these conditions.The above data strongly indicate that N-sulfoöxy-2-aminofluoreneis the major ultimate electrophilic and carcinogenic metaboliteof N-hydroxy-AAF in the livers of infant male B6C3F₁ mice.