Journal of NeurochemistryRaven Press, Ltd ., New York© 1995 International Society for Neurochemistry

Tellurite Specifically Affects Squalene Epoxidase :Investigations Examining the Mechanism of

Tellurium-Induced Neuropathy

Maria Wagner, Arrel D . Toews, and Pierre Morell

Department of Biochemistry and Biophysics, and Brain and Development Research Center, University of North Carolina,Chapel Hill, North Carolina, U.S.A .

Abstract : A peripheral neuropathy characterized by atransient demyelinatiog/remyelinating sequence resultswhen young rats are fed a tellurium-containing diet . Theneuropathy occurs secondary to a systemic block in cho-lesterol synthesis. Squalene accumulation suggested thelesion was at the level of squalene epoxidase, a micro-somal monooxygenase that uses NADPH cytochromeP450 reductase to receive its necessary reducing equiva-lents from NADPH . We have now demonstrated directlyspecificity for squalene epoxidase; our in vitro studiesshow that squalene epoxidase is inhibited 50% in thepresence of 5 pM tellurite, the presumptive in vivo activemetabolite . Under these conditions, the activities of othermonooxygenases, aniline hydroxylase and benzo(a)pyr-ene hydroxylase, were inhibited less than 5%. We alsopresent data suggesting that tellurite inhibits squaleneepoxidation by interacting with highly susceptible -SHgroups present on this monooxygenase. In vivo studiesof specificity were based on the compensatory responseto feeding of tellurium. Following tellurium intoxication,there was up-regulation of squalene epoxidase activityboth in liver (11-fold) and sciatic nerve (fivefold) . Thisinduction was a specific response, as demonstrated inliver by the lack of up-regulation following exposure tothe nonspecific microsomal enzyme inducer, Phenobarbi-tal . As a control, we also measured the microsomalmonooxygenase activities of aniline hydroxylase andbenzo(a)pyrene hydroxylase . Although they were in-duced following Phenobarbital exposure, activities ofthese monooxygenases were not affected followingtellurium intoxication, providing further evidence of speci-ficity of tellurium intoxication for squalene epoxidase.Key Words: Tellurium-Tellurite-Neuropathy-Squa-lene epoxidase-Cholesterol .J. Neurochem. 64, 2169-2176 (1995) .

A transient peripheral neuropathy associated with ahighly synchronous primary demyelination of the sci-atic nerves results when weanling rats are fed a tellu-rium-containing diet (Lampert et al ., 1970 ; Lampertand Garrett, 1971 ; Duckett et al ., 1979 ; Said et al.,1981 ; Takahashi, 1981) . The peripheral demyelination

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is secondary to the tellurium-induced metabolic blockof cholesterol synthesis (Harry et al ., 1989 ; Wagner-Recio et al ., 1991) . The lack of cholesterol, a promi-nent component of myelin and one with heavy respon-sibility for maintenance of the specialized structuralcharacteristics of this membrane, leads to destabiliza-tion and collapse of this structure .

In the tissues of rats fed tellurium, radioactive tracersentering the sterol synthesis pathway accumulate insqualene. We therefore proposed the metabolic blockto be at the enzyme that catalyzes the conversion ofsqualene to squalene 2,3-oxide (squalene epoxidase ;EC 1 .1 .1 .34), the first oxygen-dependent step in cho-lesterol biosynthesis . This microsomal enzymatic sys-tem is comprised of NADPH cytochrome P450 reduc-tase with squalene epoxidase as the terminal oxidase(Ono et al ., 1980) . In addition to the microsomal com-plex, a cytosolic sterol carrier protein (Srikantaiah etal ., 1976), as well as FAD, NADPH, and molecularoxygen, is required for activity .

In the present study, we provide further evidencefor the highly specific in vivo inhibition of squaleneepoxidase subsequent to feeding of tellurium . This re-markable specificity also can be demonstrated in cell-free preparations with micromolar concentrations oftellurite (Te03) -2 , suggesting this is the active metab-olite in vivo . These data, along with information ob-tained previously, give insight into the reason why,subsequent to tellurium feeding, cholesterol synthesisin the PNS is affected (resulting in demyelination) atthe same time that normal levels of cholesterol synthe-sis are maintained in liver .

Received July 13, 1994 ; revised manuscript received October 21,1994 ; accepted October 25, 1994 .

Address correspondence and reprint requests to Dr . P . Morell atBDRC CB# 7250, University of North Carolina, Chapel Hill, NC27599-7250, U.S.A .

Abbreviations used: NEM, N-ethylmaleimide ; HMG-CoA reduc-tase, 3-hydroxy-3-methylglutaryl-CoA reductase .

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MATERIALS AND METHODS

MaterialsSolvents were HPLC-grade and obtained from Fisher Sci-

entific (Fair Lawn, NJ, U.S.A .), and reagent-grade chemicalswere obtained from Sigma (St. Louis, MO, U.S.A.), unlessotherwise noted.

Animal modelsAll animal use procedures were in strict accordance with

the NIH Guide for the Care and Use ofLaboratory Animalsand were approved by the local Animal Care Committee.The procedure for exposure of animals to tellurium has beendescribed previously (Wagner-Redo et al ., 1991) . In brief,20-day-old male Long-Evans rats were placed on a tellu-rium-containing diet for 3 days . Preparation of the diet in-volved addition of elemental tellurium powder (60 mesh ;Aldrich, Milwaukee, WI, U.S.A .) to milled Purina rodentchow to give a final concentration of 1.1% tellurium byweight, with corn oil (12% final concentration) added toprevent separation of the mixture. Control animals weremaintained on a regular milled chow and corn oil diet . Waterwas provided ad libitum . In other experiments, 20-day-oldLong-Evans rats were injected intraperitoneally for twoconsecutive days with 70 mg/kg sodium phenobarbitalfreshly dissolved in normal saline and were killed on day 3 ;controls received saline alone.Following exposure to toxicant, animals were anesthetized

with ether and killed by decapitation . Sciatic nerves weredissected and desheathed prior to subcellular fractionation .When liver was used for preparation of microsomes, theposterior vena cava was ligated, and the liver perfused insitu via the portal vein with 30 ml of cold 0.1 M Tris-HCl(pH 7 .5 at 4°C) . All further steps were performed at 4°C.

Subcellular fractionationLiver microsomes were prepared according to the proce-

dure of Yamamoto and Bloch (1970) with some modifica-tions. Livers were minced and homogenized using a loose-fitting Teflon pestle in a Potter-Elvehjem homogenizer withtwo volumes of 0.1 MTris-HCl (pH 7.5 ) at 4°C. The homog-enate was centrifuged at 10,000 g for 10 min, and the floatinglipid layer was discarded. The supernatant was removed us-ing a Pasteur pipette and then centrifuged at 95,000 g for90 min. The resulting supernatant was discarded and themicrosomal pellet was washed twice with 0.1 M Tris-HClat pH 7.5 and resuspended in this buffer at one-third theoriginal liver weight. Microsomes (at a yield of 5.6 mg ofprotein/g wet weight liver) were stored at -70°C until use.For preparation of sciatic nerve microsomes, three pairs of

desheathed nerves were homogenized at 4°C using a Kontesground-glass pestle and homogenizer, in a total of 2 ml of0.1 M Tris-HCl at pH 7 .5 . The remainder of the isolationwas performed as for liver, with the resulting sciatic nervemicrosomes being resuspended in 500 ~l of the same bufferand stored at -70°C until use (yield of 0.40 mg per threepairs of desheathed sciatic nerves from 23-day-old animals) .Protein was determined using the Bio-Rad protein assay re-agent (Hercules, CA, U.S.A .) with bovine serum albuminas standard .

Preparation of [' °C] squaleneSciatic nerves from tellurium-exposed animals were incu-

batedwith [1-"C]acetate (ICN Radiochemicals, Irvine, CA,U.S.A .), and radioactive squalene was extracted for use inthe in vitro squalene epoxidase assay, as described pre-

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viously (Wagner-Redo et al ., 1991), with the followingmodifications. Instead of preparative TLC, the lipid extractwas loaded onto silica Sep-Pak cartridge (Marlborough, MA,U.S.A .) preequilibrated with heptane/benzene (90:10), andradioactive squalene was eluted by gravity . After evapora-tion of solvent, squalene was resuspended in chloroform/methanol (1 :1) and a sample was injected and analyzed forpurity by reverse-phase HPLC using an isocratic gradientof acetonitrile/isopropanol (90:10) ; see Chromatographicprocedures) . Eluted radioactivity was determined followingthe addition of ScintiSafe Econo 1 (Fisher Scientific) usingan LKB Rackbeta Counter. Greater than 98% of total radio-activity in this sample colocalized with the squalene peak .

Squalene epoxidase assayIt is, in principle, possible to carry out this assay with

microsomes as the source of enzyme (cytochrome P450 re-ductase and squalene epoxidase), and cytosol as the sourceof the carrier protein, which is required for presentation ofsqualene. This assay is not, however, suitable for study ofinhibition of the reaction by tellurite, as other cytosolic en-zyme activities include a coupling factor that transfers elec-trons from NADPH to tellurite (Te03) -2 , converting it toelemental tellurium (indicated by precipitation of the insolu-ble element) .To eliminate this interfering activity, we used the proce-

dure of Ono and Bloch (1975) in which the nonionic deter-gent, Triton X-100, replaces the requirement for the sterolcarrier protein located in the cytosol. The detergent exposesthe membrane-embedded squalene epoxidase complex, mak-ing it available to interact with its substrate. This assay,therefore, permitted in vitro studies of squalene epoxidaseactivity using tellurite concentration ranges reflective of anin vivo poison .A dispersion of ['°C]squalene (25,000 dpm, 8 ~M final

concentration) was prepared in a 16 X 125-mm borosilicateglass tube with 40 ~1 of 0.1% Tween 80 in acetone. Theacetone was removed by a stream of nitrogen, and the sub-strate dispersed in 0.1 MTris-HCl (pH 7.5 at 37°C) and 100~1 of 0.5% Triton X-100 (electrophoresis grade, FisherBio-tech, Fair Lawn, NJ, U.S.A.) using a vortex mixer followedby agitation in a sonicating water bath for 1 min. The assaymixture also contained 0.01 mM FAD, 1 mM NADPH, 1mM EDTA, and 200 Fig of microsomal protein in 500 ~1 of0.1 M Tris-HCl (pH 7.5 at 37°C) . When indicated, telluriteas potassium tellurite, or N-ethylmaleimide (NEM) was dis-solved in the same buffer and added to the incubation atindicated concentrations, Incubations were for 20 min at37°C in a metabolic shaking water bath and were terminatedby the addition of 500 ~1 of 10% KOH in methanol . Sampleswere capped and, following saponification at 80°C for 60min, lipids were extracted using a modification (Benjaminset al ., 1976) of the method of Folch et al . (1957 ) . Thesolvent layer was evaporated under nitrogen, and lipids weresubjected to either TLC or reverse-phase HPLC . The assaywas linear to a protein concentration of 1 mg and, at 200Ng, with time up to 60 min.

Chromatographic analysis for measuring squaleneepoxidase activityWhen measuring the in vitro effect of tellurite or NEM on

squalene epoxidase, it was sufficient to separate the enzymeproducts, squalene 2,3-oxide and small amounts of lanos-terol, from the radioactive substrate by TLC on 0.25-mmsilica gel GHL plates (Analtech, Newark, DE, U.S.A .) pre-

INHIBITION OF SQUALENE EPOXIDASE BY TELLURITE

developed in chloroform/methanol (4:1) . Samples werespotted with standards as carriers and separated using a sol-vent system of benzene/acetone (90:1) . Lipids were visual-ized by iodine vapor, and the area corresponding to eachlipid was scraped into a scintillation vial . The silica gel wasdeactivated with 0.8 ml of water, and 5 ml of ScintiSafeEcono 1 (Fisher Scientific) was added . Radioactivity wasdetermined using an LKB Rackbeta Counter .

For assays of enzyme activity in microsomas from animalstreated in vivo, there was a complication ; accumulation ofendogenous squalene (ordinarily present only at very lowlevels ) diluted the specific radioactivity of the radioactivesqualene added as substrate . Therefore, in these assays, anal-ysis of lipids to determine squalene epoxidase activity wasdone by reverse-phase HPLC . This method allowed for cor-rection of the dilution by endogenous squalene of specificradioactivity of the added [ "C] squalene . The products,squalene 2,3-oxide and sterols, were separated from squaleneby reverse-phase HPLC on a C18 column (LiChosorb RP-18, 10 gym, 200 X 4.6 mm; Hewlett-Packard, Kennet Square,PA, U.S.A .) . The liquid chromatography equipment con-sisted of a Model IIIG Constametric pump (LDC Analytical,Riviera Beach, FL, U.S.A .), a Model 7125 loading sampleinjector (Rheodyne Inc ., Cotati, CA, U.S .A .), and a one-pump gradient controller (Autochrom Inc ., Milford, MA,U.S.A .) . The absorbance detector was a mode1500 VariableWavelength Detector, Scientific Systems Inc ., State College,PA, U.S.A .) . Lipids were eluted isocratically in 30 min at aflow rate of 2 ml/min with acetonitrile/isopropanol (90:10)and detected by absorbance at 210 nm . After sample elution,the column was washed for 25 min with isopropyl/methanol(70:30) prior to loading a new sample . The UV detectorwas connected to an on-line integrator (Nelson Analytical900 Series Interface, Cupertino, CA, U.S.A.) for analysis ofsqualene peak areas based on a squalene standard curve .Eluate fractions of 60-s duration were collected . Fractionswere dried using a stream of nitrogen, 5 ml of ScintiSafeEcono 1 was added, and the radioactivity was determined.

Other assaysActivity of benzo(a)pyrene hydroxylase was determined

by the conversion of [ 3H]benzo(a)pyrene (70 Ci/mmol, Am-ersham, Arlington Heights, IL, U.S.A .) to aqueous-solubleproducts, according to the procedure ofDePierre et al . (1978 ) .When indicated, tellurite, as the potassium salt, was added tothe desired concentration from stock solutions prepared in theassay buffer. The assay used to measure aniline hydroxylaseactivity was a modification (O'Brien and Rahimtula, 1978)of the spectrophotometric assay of Imai et al. (1966) . Whenindicated, potassium tellurite was added as described above .NADPH cytochrome P450 reductase activity was measuredusing exogenous cytochrome c as receptor (Hrycay et al .,1975 ) , using an Hitachi U-2000 Series temperature-controlledspectrophotometer with accompanying software . Activity wascalculated using the following formula: nanomoles of cyto-chrome c reduced per minute = DAsso/min/0 .021 . Theamount of microsomal protein used was such that the initialO Also value did not exceed 0.2 min .- '

RESULTS

In vitro, tellurite specifically inhibits themonooxygenase, squalene epoxidaseThe specificity of tellurite inhibition of squalene

epoxidase was tested by comparison with its action on

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FIG . 1 . Effect of tellurite on the activity of monooxygenases .Microsomes were prepared from liver of 21-day-old control rats,and activity of each monooxygenase was measured in the ab-sence and presence of tellurite, as described in Materials andMethods . Results are expressed as percentage of the activity inthe absence of tellurite . Individual points represent the meansSEM of duplicate determinations ; n = 4 separate experiments

for aniline hydroxylase, and n = 10 for squalene epoxidase . Theassay for benzo(a)pyrene hydroxylase was a single experiment,with points representing the means of triplicate determinations .

very similar enzymes. Because squalene epoxidase isa microsomal cytochrome P450 reductase-linked ter-minal oxidase, we chose as controls other microsomalcytochrome P450 reductase-linked terminal oxi-dases-the xenobiotic-transforming enzymes anilinehydroxylase and benzo(a)pyrene hydroxylase (Yangand Lu, 1987) . Although the tissue of initial interestwith respect to pathology induced by tellurium wassciatic nerve (because of the observed demyelination),these control enzymes are not present in nerve . Thus,the enzyme source for the assay was liver of weanling21-day-old control rats . Squalene epoxidase activitywas half inhibited in the concentration range of 5-10 IcM tellurite, reasonable for an in vivo metabolictoxicant (Fig . 1) . In contrast, both aniline hydroxylaseand benzo(a)pyrene hydroxylase activities were in-hibited less than 5% within this concentration range .Although not shown, additional in vitro studies wereperformed measuring the activity of NADPH cyto-chrome P450 reductase ; even at a concentration of 10mM tellurite, reductase activity was not inhibited .We suggest tellurite acts as a reversible inhibitor of

squalene epoxidase . Evidence in support of this con-clusion was obtained by checking for time dependenceof the preineubation with tellurite . At 5 ~M tellurite,both 2- or 20-min preincubations resulted in the same50% inhibition .

Evidence for specificity of the effects of telluriumfeeding in vivo

Testing for specificity of tellurium intoxication invivo involved assay of compensatory responses . Thehypothesis was that there are, for the three enzymes,independent compensatory responses of up-regulationsubsequent to inhibition . If this were so, the telluriumfeeding should bring about a compensatory response

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FIG . 2 . In vivo response of squalene epoxidase activity in sciaticnerve and liver following exposure to tellurium (Te) or phenobar-bital (Phe) . Rats, 20 days of age, were either placed on a dietcontaining 1 .1 % tellurium for 3 days, or given daily intraperito-neal injections of 70 mg/kg phenobarbital for 2 days, and squa-lene epoxidase activity was measured in microsomal prepara-tions as described in Materials and Methods . Individual pointsrepresent the means ± SEM of duplicate determinations withineach experiment . The number of preparations of pooled nervesamples was n = 5 for control (C), n = 3 for tellurium-fed ani-mals, and n = 2 for phenobarbital-injected animals . For liver, n= 12 for control (similar values for both control groups plottedas one value), n = 6 for tellurium-fed animals, and n = 6 forphenobarbital-injected animals .

specifically in squalene epoxidase . The observed resultwas clearcut ; 3 days of tellurium feeding produced amarked up-regulation of squalene epoxidase activityin both liver (Fig . 2, lower panel) and sciatic nerve( Fig . 2, upper panel ) . The up-regulation was greaterin liver (11-fold) than in nerve (fivefold) . In contrast,tellurium feeding had no effect on either of the othertwo microsomal monooxygenases examined, anilinehydroxylase and benzo(a)pyrene hydroxylase (Fig .3), providing further evidence of specificity of tellu-rium for squalene epoxidase .

It is well known that in response to generalized met-abolic insults, such as alcohol, there is proliferation ofendoplasmic reticulum (Liu et al., 1975), and micro-somal enzyme activities may increase secondary to thisproliferation . We used phenobarbital, a compound alsoknown to give such a response, as a control for thispossibility. As is evident from Fig . 2, challenge withphenobarbital did not induce squalene epoxidase activ-ity . The converse required control is to demonstratethat the same agent could up-regulate the control en-zyme activities without up-regulating squalene epoxi-dase . Indeed, treatment with phenobarbital in vivo up-regulated aniline and benzo(a)pyrene hydroxylase( Fig . 3, upper and Lower panels ) .

Experiments measuring the activity of NADPH cy-tochrome P450 reductase, the common electron carrier

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for microsomal monooxygenases, showed telluriumfeeding did not significantly affect in vivo reductaseactivity . However, as expected, this activity was in-creased following phenobarbital treatment (from 187± 1 .1 to 287 ± 7.1 nmol of cytochrome c reduced/mg of protein/min) . Another control was to measurethe in vivo response of the monooxygenase phenylala-nine hydroxylase, a monooxygenase that is located inthe cytosolic fraction and uses tetrahydrobiopterin toreceive its necessary reducing equivalents ; again, tellu-rium had no effect on this activity (data not shown ) .

In vitro, squalene epoxidase activity in sciaticnerve is somewhat more sensitive than liverto telluriteWashed microsomes were isolated from the sciatic

nerves and livers of 21-day-old control rats, and squa-lene epoxidase activity was measured in the presenceof three concentrations of tellurite . Squalene epoxidaseactivity in both sciatic nerve and liver was significantlyinhibited in the presence of low concentrations of tellu-rite (Fig . 4) . At a given tellurite concentration, how-ever, squalene epoxidase activity in sciatic nerve mi-crosomes was 30-40% more inhibited than in livermicrosomes . Whether this occurs in vivo or is an arti-fact of the in vitro assay is debatable ( see below) .

FIG . 3 . In vivo response of liver aniline hydroxylase and benzo-(a)pyrene hydroxylase activity following exposure to tellurium(Te) or phenobarbital (Phe) . Rats were exposed to either tellu-rium or phenobarbital as described in Fig . 2, and enzyme activi-ties of thesetwo monooxygenases were measured in liver micro-somes as described in Materials and Methods . Data representthe means ± SEM of duplicate determinations within each exper-iment. For aniline hydroxylase, n = 22 for control (C ; similarvalues for both control groups plotted as one value), n = 13for tellurium-fed animals, and n = 13 for phenobarbital-injectedanimals . For benzo(a)pyrene hydroxylase, n = 17 for control, n= 11 for tellurium-fed animals, and n = 16 for phenobarbital-injected animals .

INHIBITION OF SQUALENE EPOXIDASE BY TELLURITE

FIG. 4, Effect of tellurite on the activity of squalene epoxidaseactivity in sciatic nerve and liver . Microsomes were preparedfrom sciatic nerve and liver from weanling 21-day-old controlrats, and squalene epoxidase activity was measured in the pres-ence of three tellurite concentrations, as described in Materialsand Methods. Results are expressed as percentage activity inthe absence of tellurite . Individual points represent meansSEM of three separate experiments.

Squalene epoxidase may contain -SH groupssusceptible to modification

Tellurite has been suggested to interact with -SHgroups on or near the surface of some enzymes andresult in the inhibition of their activity (Wachstein,1949 ; Siliprandi et al ., 1971) . We therefore investi-gated the possibility of squalene epoxidase containingsusceptible -SH groups by measuring squalene epoxi-dase activity in the presence of the known sulfhydrylreagent NEM (Webb, 1966) . The results in Fig . 5show that squalene epoxidase activity declines sharplyin the presence of NEM.

Because these results suggested that squalene epoxi-dase possessed susceptible -SH groups that were neces-sary for enzyme activity, we also examined the effect

FIG. 5. Effect of NEM on squalene epoxidase and aniline hy-droxylase activity. Washed liver microsomes were prepared from21-day-old rats, and enzyme activities in the presence of variousconcentrations of the sulfhydryl reagent NEM were measuredas described in Materials and Methods. Results are expressedas the percentage of enzyme activity in the absence of NEM.Values for squalene epoxidase activity are the means ± SEM ofduplicate determinations in four separate experiments, and thevalues for aniline hydroxylase are the means of duplicate deter-minations in which the mean differed by less than 5% for eachconcentration .

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FIG. 6. Kinetic analysis of tellurite's effects on squalene epoxi-dase activity with respect to various squalene or FADconcentra-tions. In vitro squalene epoxidase assays were performed usingliver microsomes prepared from 21-day-old control rats. Line-weaver-Bunk analysis of squalene epoxidase activity in the pres-ence of 5 ~cM tellurite shows a noncompetitive inhibition withrespect to squalene and a mixed-type inhibition with respect toFAD.

of NEM on the monooxygenase aniline hydroxylase .Similar to the results observed with tellurite (Fig . 1),NEM had little effect on aniline hydroxylase activitycompared with squalene epoxidase activity (Fig . 5) .

Tellurite may inhibit squalene epoxidase activityby more than one mechanism

Presumably, at the enzyme level, tellurite inhibitssqualene epoxidase in a similar fashion in both sciaticnerve and liver . Due to the abundance of microsomelprotein available from liver, the following studies wereperformed using washed microsomes isolated fromcontrol livers. Squalene epoxidase activity was mea-sured in the absence and presence of a fixed (5 ~M)concentration of tellurite while the concentration ofthe substrate, squalene, or the cofactor, FAD, waschanged . Lineweaver-Bunk analyses (Segel, 1976) ofexperiments performed using various squalene concen-trations in the presence of 5 ~M tellurite show a non-competitive type of inhibition, with a mutual Km valueof approximately 4 ~M (Fig . 6, upper panel) . Similaranalyses of experiments using 5 ~M tellurite and vari-ous concentrations of FAD show that tellurite exhibitsa "mixed-type" inhibition (Fig . 6, lower panel) . The

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data for FAD dependence in the presence of telluritecould fit a hyperbolic curve, suggesting the possibilityof a two-site model for binding of FAD. Such a plot,however, resulted in a negative Km value, making thishypothesis unlikely .

DISCUSSION

We have tested our hypothesis that the metaboliclesion induced by feeding of tellurium is highly spe-cific, resulting in an inhibition of squalene epoxidaseactivity . This suggestion arose because, subsequent tofeeding of tellurium, there is accumulation of squaleneand, to a variable extent in different tissues, inhibitionof cholesterol formation . The strategies to test the hy-pothesis are critically dependent on proper choice ofcontrol enzymes . Squalene epoxidase is a monooxy-genase receiving electrons from NADPH via cy-tochrome P450 reductase . We, therefore, selected ascontrols two other monooxygenases that are terminaloxidases for cytochrome P450 reductase-aniline hy-droxylase and benzo (a) pyrene hydroxylase .The suggestion that tellurite is the active metabolic

inhibitor arose from the demonstration that when radio-active precursors of cholesterol were incubated withsciatic nerve segments or liver slices in the presenceof tellurite, label accumulated in squalene (Wagner-Recio et al ., 1991) . Preliminary studies with a postmi-tochondrial supernatant supported the idea that telluriteinhibits squalene epoxidase . This crude assay is notquantitative, due to the presence of a factor in crudehomogenate that directly couples transfer of electronsfrom NADPH to tellurite, causing formation and pre-cipitation of insoluble tellurium . The assay used inthe present study substituted a low concentration ofdetergent for the otherwise obligatory squalene carrierprotein found in the supernatant . We demonstrated that5 ~,M tellurite, a concentration that may reasonablyaccumulate in tissue under these circumstances, halfinhibited squalene epoxidase activity ; the control en-zymes were not affected until much higher concentra-tions of tellurite were used .An extension of the in vitro studies addressed the

possibility that squalene epoxidase is inhibited throughthe action of tellurite as a reversible sulfhydryl reagent( Siliprandi et al ., 1971 ; De Meio and Doughty, 1979) .This is analogous to the more extensively studied inter-actions of selenium with -SH groups (Sandholm andSipponen, 1973 ; Young et al ., 1981) . Our results withthe known sulfhydryl reagent NEM demonstrated thatsqualene epoxidase has susceptible -SH group (s) , andthat these groups) are necessary for activity . The ki-netics of inhibition of enzyme activity with tellurite(reversible inhibitor) are not directly comparable withthose obtained with NEM (irreversible inhibitor) .NEM is, however, generally considered to be a highlyspecific sulfhydryl reagent that may have selectivityfor certain cysteines within a particular protein (Webb,1966) . The sensitivity of squalene epoxidase sulfhy-

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dryl groups to NEM, at micromolar concentrations,is quite marked relative to millimolar concentrationsrequired to inhibit many other enzyme activities(Webb, 1966) . In contrast, aniline hydroxylase wasnot inhibited within the same concentration range ofNEM. We note a passing comment by Ryder and Du-pont (1984) that 1 mM NEM inhibits yeast squaleneepoxidase activity by about 30% . The mammalian en-zyme, susceptible to NEM concentrations more thantwo orders of magnitude lower, may have differentstructural characteristics .The strategy for testing the metabolic block in vivo

assumed that squalene epoxidase is feedback regulatedby its product, squalene epoxide, or some further me-tabolite, such as cholesterol (Edenberg and Schechter,1984 ; Hidaka et al ., 1990 ; Sato et al., 1990) . Thus,up-regulation of squalene epoxidase should occur con-sequent to tellurium feeding . This indeed was the case .Specificity was demonstrated, as aniline hydroxylaseand benzo (a ) pyrene hydroxylase were not up-regu-lated . A control experiment involved animals treatedwith phenobarbital, to show that benzo(a ) pyrene andaniline hydroxylase and the mutually shared cyto-chrome P450 reductase could be up-regulated; this oc-curred under conditions in which squalene epoxidaseactivity was not affected . Neither tellurium nor pheno-barbital had nonspecific effects mediating oxygenasesin general as neither affected phenylalanine hydroxy-lase activity .

Susceptibility of squalene epoxidase to telluriumwas greater in nerve than in liver when examined invitro . This, however, does not seem sufficient to ac-count for the much greater in vivo susceptibility ofnerve to tellurium toxicity and, in fact, we do not knowhow meaningful the 30% difference observed is . Al-though reproducible, it could be an artifact of the assayprocedure, i .e ., homogenization ofthe lipid-rich sciaticnerve may have created a squalene epoxidase-mem-brane complex which, after treatment with detergentin the assay mixture, presents squalene epoxidase in aconfiguration more susceptible to inhibition by tellu-rite . Another factor relevant to tissue specificity is thatalthough the in vivo response to tellurium challengeinvolves up-regulation of squalene epoxidase activity,up-regulation is twice as great in liver as in sciaticnerve . Nevertheless, it seems that neither preferentialsusceptibility of nerve squalene epoxidase to telluritein vitro nor the greater in vivo up-regulation of squa-lene epoxidase in liver relative to nerve is sufficientto account for the quantitative observation (Wagner-Recio et al ., 1991) that tellurium feeding causes greatinhibition of cholesterol synthesis in nerve, while cho-lesterol output by liver is maintained at normal levels .

In view of these results, we suggest that the explana-tion as to why liver is somewhat protected againsttellurium challenge does not relate primarily to proper-ties of the immediate target, squalene epoxidase, butrather has to do with the previously reported (Toewset al ., 1991) tissue-specific responses to tellurium feed-

INHIBITION OF SQUALENE EPOXIDASE BY TELLURITE

ing of 3-hydroxy-3-methylglutaryl-CoA reductase(HMG-CoA reductase ) , the rate-limiting enzyme ofcholesterol biosynthesis . Following tellurium intoxica-tion, liver HMG-CoA reductase is up-regulated by al-most an order of magnitude, resulting in massive accu-mulation of squalene. We suggest that the squaleneepoxidase reaction is driven by mass action . In con-trast, HMG-CoA reductase is down-regulated in sciaticnerve (Toews et al ., 1991), minimizing buildup ofsqualene .

In general, it is not surprising that control of choles-terol synthesis would be different in liver and nerve .The liver maintains an output of very low density lipo-proteins for delivery of energy substrates as triglycer-ides to the periphery, and the supply of cholesterol forthese lipoproteins must be tightly regulated (Dietschyet al ., 1993 ) . The activity ofliver HMG-CoA reductaseresponds rapidly to changes in levels of circulatingcholesterol . In contrast, cholesterol synthesis inSchwann cells is related primarily to the need for my-elination, which presumably is programmed accordingto interaction of cells within nerve . The control oftranscription of HMG-CoA reductase in sciatic nerveinvolves genomic-level control factors (transcriptionfactors, enhancers), which regulate transcription ofHMG-CoA reductase in parallel with genes inducedfor myelination . We have shown that in Schwann cells,the initial tellurium-induced block in synthesis of cho-lesterol rapidly results in reduction of levels of mRNAfor myelin proteins (Toews et al ., 1990) . As expected,synthesis of cholesterol by nerve does not respond tochanges in dietary cholesterol ; in fact, sciatic nervedoes not have access to circulating cholesterol (Jure-vics and Morell, 1994) .We suggest that the preferential vulnerability of my-

elin formation to tellurium toxicity relates to genomic-level differences between glial cells and liver with re-spect to control of cholesterol biosynthesis, rather thanto tissue specificity in metabolism of tellurium or com-partmentalization of metabolites . Specificity for thePNS, rather than the CNS, is a function of the muchgreater rate of accumulation of myelin ( and consequentdemand for cholesterol ) of sciatic nerve relative toCNS (Rawlins and Smith, 1971) .

Acknowledgment : We thank Dr . Michael D . Robersontor helping to desheath the sciatic nerves for the experiments,and Janice E . Weaver and Haven A . Conley for their excel-lent care and management of animals . This study was sup-ported by USPHS grants ES-01104 and NS-11615 .

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