Relative merits of immunohistochemical demonstrations of placental, A, B and C forms of glutathione S-transferase and histochemical demonstration of {gamma}-glutamyl transferase as markers of altered foci during liver carcinogenesis in rats

The values of the immunohistochemical demonstrations of glutathioneS-transferases (GSTs) A, B, C and P and histo-chemical demonstrationsof -glutamyl transpeptidase (-GT) for detection of enzyme alteredfoci in F344 rat liver were compared. Rats were given a singlei.p. injection of 200 mg/kg body weight of diethylnitrosamine(DENA), from 2 weeks later they were given 0.02% N-2-fluorenylacetamide(2-FAA), phenobarbital (PB), butylated hydroxyanisole (BHA)or butyl-ated hydroxytoluene (BHT) in their diet for 6 weeksand then they were given basal diet and tap water for 4 weeks.They were subjected to partial hepatectomy at the end of week3. Results showed that immunohistochemical demonstration ofGSTs A, B and C for detection of foci were only effective whenthe administration of 2-FAA, PB, BHA or BHT in the diet wasdiscontinued, because these GSTs were induced in surroundinghepatocytes by these compounds in the diet. -GT was inducedin periportal hepatocytes strongly by BHA and BHT and slightlyby PB, and -GT positive foci in periportal areas were not distinguishablefrom -GT positive peri-portal hepatocytes. GST-P was also inducedmoderately by BHA and slightly by BHT in periportal hepatocytes,but all GST-P positive foci were clearly distinguishable. Inaddition, almost all -GT positive foci gave a positive reactionfor GST-P, but 5–10% of the GST-P positive foci were not-GT positive.     

Modification by antioxidants and p, p'-diaminodiphenylmethane of 7, 12-dimethylbenz[a]anthracene-induced carcinogenesis of the mammary gland and ear duct in CD rats

The effects of antioxidants on mammary gland carcinogenesispretreated with 7, 12-dimethylbenz[a]anthracene (DMBA) in femaleSprague-Dawley rats were examined. The antioxidants used werebutylated hydroxyanisole (BHA), butylated hydr-oxytoluene (BHT),sodium L-ascorbate, -tocopherol, ethoxy-uin and p, p’-diaminodiphenylmethane(DDPM), which is an inhibitor of carcinogenesis in the liver,kidney and urinary bladder. Female Sprague-Dawley rats of 50days old were treated with 2.5 mg/100 g body weight of DMBA,and from 1 week later were given diet supplemented with 1% BHA,0.7% BHT, 5% sodium L-ascorbate, 1.5% -tocopherol, 0.5% ethoxyquinor 0.1% DDPM for 33 weeks and then killed. The incidences ofmammary tumors, carcinomas and fibroaden-omas in DMBA-treatedanimals were reduced by diet containing BHA or ethoxyquin. Dietcontaining BHT or DDPM inhibited the induction of only fibroadenomas.The incidence of ear duct tumors in DMBA-treated animals wasreduced by diet containing BHT, -tocopherol or ethoxyquin.     

Modifying effects of butylated hydroxyanisole, ethoxyquin and acetaminophen on induction of neoplastic lesions in rat liver and kidney initiated by N-ethyl-N-hydroxyethylnitrosamine

Studies were made on the effects of butylated hydroxyanisole(BHA), ethoxyquin (EQ) and acetaminophen (AAP) on the inductionof neoplastic lesions in the liver and kidney of rats initiatedby N-ethyl-N-hydroxyethylnitrosamine (EHEN). The number andarea of histochemical -glutamyltrans-peptidase-positive (-GT⁺)foci per unit area of liver section in rats given BHA, EQ orAAP were significantly less than in rats given EHEN alone. Similarly,the number of hyperplastic nodules (HN) in groups given BHAor AAP and their area in groups given BHA, EQ or AAP were significantlyless than in control groups. Induction of hepatocellular carcinoma(HCC) was also clearly inhibited by these three chemicals. Noliver lesions were found in any animals given BHA, EQ or AAPorally without EHEN. In contrast, the incidence and quantitativevalues of preneoplastic lesions and renal cell adenoma weresignificantly increased in groups given BHA, EQ or AAP. Theresults clearly demonstrated that BHA, EQ and AAP inhibitedthe development of -GT⁺ foci, HN and HCC, whereas they enhancedthe appearance of preneoplastic and neoplastic lesions in thekidney.     

Induction by butylated hydroxyanisole of specific molecular forms of glutathione S-transferase and UDP-glucuronyltransferase and inhibition of development of {gamma}-glutamyl transpeptidase-positive foci in rat liver

Effects of an antioxidant, butylated (3-tert-butyl-4-) hydroxyanisole(BHA) on the induction of specific molecular forms of glutathioneS-transferase (GST), UDP-glucuronyltransferase (UDP-GT) andother glutathione-related enzymes in rat liver were investigated.The development of -glutamyl transpeptidase (-GTP)-positivefoci and hyperplastic nodules induced by diethylnitrosamine,200 mg/kg i.p., followed by 0.02% N-2-fluorenylacetamide (FAA)in diet plus partial hepatectomy was inhibited by the administrationof 0.75% BHA in the FAA-containing diet. Inhibition was reflectedin decreased area of -GTP-positive foci which correlated witha decrease in -GTP activity measured biochemically. Under thepresent experimental conditions, total activities of GSTs, especiallythat of GST-A form, and of UDP-GTs, especially that of the latefetal form (o-GT), were markedly increased, together with glutathionelevels in the whole liver, within one week after BHA administration.Without BHA administration the activities of GST-A and o-GT,as well as glutathione levels, were also increased by FAA treatment,primarily localized within -GTP-positive foci. These resultssuggest that the induction of specific molecular forms of detoxicatingenzymes either in enzyme-altered foci or in the whole livermay play an important role in determining the extent of developmentof preneoplastic nodules from initiated foci under the shortterm induction conditions used.     

Enhancement of BHA-induced proliferative rat forestomach lesion development by simultaneous treatment with other antioxidants

Synergistic effects of butylated hydroxyanisole (BHA) and otherantioxidants on induction of rat forestomach lesions were investigated.Groups of F344 male rats were treated with 1% BHA phis 0.7%butylated hydroxytoluene (BHT), 1% BHA phis 1% propyl gallate(PG), 1% BHA plus 1% sodium L-ascorbate (SA), 1% BHA phis 1%DL--tocopherol (-TP), 0.4% BHT phis 0.4% BHA plus 0.4% PG plus0.4% SA plus 0.4% -TP, 1% BHA or 2% BHA. Further groups of 10rats each received antioxidants without BHA as controls. Histo-togfcalexamination revealed significantly increased incidences of hyperplasiain the groups given BHA together with SA or PG at the prefundicregion or at the mid region respectively. The forestomach changesinduced by BHA together with SA were equal to those inducedby 2% BHA. On the other hand, simultaneous treatment with BHAand PG or -TP reduced the incidence of hyperplasia at the prefundkregion. It is concluded that mixed treatment with BHA and otherantioxidants exerted enhancing or inhibitory effects on theinduction of hyperplasia at different sites of the forestomachepithelium.     

Isolation of {gamma}-glutamyl transpeptidase positive hepatocytes during the early stages of hepatocarcinogenesis in the rat

-Glutamyl transpeptidase (GT) positive hepatocytes were isolatedfrom F344 male rats fed 2-acetylaminofluorene. The isolationprocedure is rapid and highly selective for cells exhibitingGT on their surface. Suspensions of liver cells obtained fromperfusion in situ with a collagenase solution were incubatedon Petri dishes coated with affinity purified rabbit anti-GTantibody. GT-positive cells bound to the dish within fifteenminutes and could be recovered as viable cells. Approximately15% of the GT-positive cells are isolated using this procedure.Novikoff hepatoma cells, a GT-positive cell line, were usedto define the parameters of the assay. The binding was bothtime and temperature dependent. Binding of cells to the anti-GTantibody coated dishes was 50% inhibited by 2.8 mM sodium azideand 86% inhibited by 4.6 mM.     

Intercellular communication in primary cultures of putative preneoplastic and 'normal' hepatocytes

Gap junctional intercellular communication (IC) was studiedin -glutamyltranspeptidase (-GT)-positive and -negative hepatocytesisolated from carcinogen-treated rats. Putative preneoplastic-GT-positive hepatocytes were visualized in monolayer culturesby indirect immunofluorescence using anti--GT-antibodies. ICwas evaluated by studying dye coupling of the cells. -GT-positivehepatocytes showed a significantly lower dye coupling than did-GT-negative liver cells. Spread of the dye Lucifer Yellow CHto neighboring cells was decreased further by the tumor-promotingchemical phenobarbital in both cell types in vitro. Also treatmentin vivo with the barbiturate significantly reduced dye couplingof hepatocytes. The findings suggest that as a result of theirdecreased ability to communicate, preneoplastic hepatocytesmay escape from growth control and differentiation signals givenout by surrounding ‘normal’ cells.     

Induction of hepatic glutathione S-transferase activity by butylated hydroxyanisole and conjugation of benzo[a]pyrene diol-epoxide

Dietary administration of 2(3)-tert-butyl-4-hydroxyanisole (BHA)to mice caused an increase in the hepatic soluble glutathioneS-transferase activity towards (±)-7ß, 8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) of 5-fold whereasthat towards 1-chloro-2,4-dinitrobenzene (CDNB) was increasedby 14-fold. Whereas with either substrate the catalytic capacityof the enzyme was elevated by BHA treatment, there was littleeffect on the K_m for CDNB but an increase in the K_m for BPDEas substrates. The results thus suggest that BHA-induced GSHS-transferase activity may be of limited importance for protectionfrom certain reactive intermediates of polycyclic aromatic hydrocarbons.     

1,N6-etheno-2' -deoxyadenosine and 3,N4-etheno-2'-deoxycytidine detected by monoclonal antibodies in lung and liver DNA of rats exposed to vinyl chloride

1,N⁶-Etheno-2'-deoxyadenosine (dAdo) and 3,N⁴-Etheno-2'-deoxycytidine(dCyd) are formed in vitro by reaction of DNA with the electrophilicmetabolites of vinyl chloride (VC), chloroethylene oxide andchloroacetaldehyde. To detect and quantitate these DNA adductsin vivo, we have raised a series of specific monoclonal antibodies(Mab). Among those, Mab EM-A-1 and Mab EM-C-1, respectively,were used for detection of dAdo and dCyd by competitive radioimmunoassay(RIA), following pre-separation of the etheno adducts from DNAhydrolysates by high perfonnance liquid chromatography. At 50%inhibition of tracer-antibody binding, both Mab had a detectionlimit of 187 fmol and antibody affinity constants (K) of 2 x10⁹ l/mol. The levels of dAdo and dyd were quantitated in theDNA of lung and liver tissue of young Sprague-Dawley rats exposedto 2000 p.p.m. of VC for 10 days. The dAdo/2'-deoxyadenosineand dCyd/2'-deoxycytidine molar ratios were 1.3 x 10^–7and 3.3 x 10^–7 respectively, in lung DNA, and 5.0 x 10^–8and 1.6 x 10^–7 in liver DNA. When hydrolysates of 3 mgof DNA were analyzed by RIA at 25% inhibition of tracer-antibodybinding, dAdo and dCyd were not detected in liver DNA from untreatedrats above the limiting dAdo/2'-deoxyadenosine and dCyd/2'-deoxycytidinemolar ratios of 2.2 x 10^–8 and 3.1 x 10^–8, respectively.     

Inhibitory effect of nafenopin upon the development of diethylnitrosamine-induced enzyme-altered foci within the rat liver

The effect of nafenopin and phenobarbitone upon the distributionof -glutamyltranspeptidase activity and epoxide hydrolase antigenicsites in the liver and upon the development of enzyme-alteredfoci during hepatocarcinogenesis have been compared. Phenobarbitoneinduced -glutamyltranspeptidase activity in perilobular hepatocytes.Nafenopin did not alter the distribution of this enzyme. Bothcompounds appeared to induce epoxide hydrolase; phenobarbitoneincreased the enzyme content of centrilobular cells, whilstnafenopin altered immunostaining mainly in portal regions. Hepaticlesions were induced by treating one day-old rats with diethylnitrosamine.Phenobarbitone and nafenopin were then administered in the dietupon weaning. Animals were killed after either 2, 4 or 8 weeksfeeding and liver sections were stained for the two enzymes.Only sections from nitrosamine-treated animals contained enzyme-alteredfoci. In general, -glutamyltranspeptidase-containing foci stainedalso for epoxide hydrolase; but many hydrolasepositive focidid not stain for -glutamyltranspeptidase activity. Phenobarbitonetreatment stimulated the formation of enzymealtered foci. Thiseffect was more marked in male animals. Nafenopin treatmentsuppressed the development of foci at all time points, suchthat less hepatic lesions were seen than in animals which receivedonly diethylnitrosamine. The results cast doubt upon the generalityof -glutamyltranspeptidase as a marker for preneoplastic lesionswithin the liver.     

Comparative histochemical investigation of the glutathione S-transferase placental form and {gamma}-glutamyltranspeptidase during N-nitrosobis(2-hydroxypropyl)amine-induced lung carcinogenesis in rats

Immunohistochemical staining using anti-rat glutathione S-transferaseplacental form (GST-P) rabbit antibody and enzyme histochemicalstaining for -glutamyltranspeptidase (-GT) were investigatedin lesions appearing during lung carcinogenesis induced by N-nitrosobis(2-hydroxypropyl)amine(BHP) in rats. Rats were given BHP at a concentration of 2000p.p.m. in drinking water, and were killed after 12 weeks ofBHP intake, after 12 weeks of BHP intake followed by 12 weeksof tap water intake or after 20 weeks of continuous BHP intake.It was found that bronchiolo-alveolar hyperplasias, adenomas,adenocarcinomas, squamous metaplasias and squamous cell carcinomashad been induced by BHP. All of the squamous metaplasias andsquamous cell carcinomas were shown to stain with GST-P butnot with -GT. On the other hand, the hyperplasias, adenomasand adenocarcinomas stained with -GT to various degrees andin different areas, but did not stain with GST-P. The incidenceof -GT phenotype and the average percentage of -GT positiveareas in hyperplasias and adenomas suggested that adenocarcinomasmight develop from hyperplasias and adenomas. These resultssuggest that GST-P is a marker for squamous lesions while -GTis a marker for adenomatous lesionsin rat lung carcinogenesis.Furthermore, squamous metaplasias appear to be preneoplasticlesions of squamous cell carcinomas while -GT-positive hyperplasiasor adenomas are preneoplastic lesions of peripheral adenocarcinomas.     

Immunohistochemical differentiation of {gamma}-glutamyltranspeptidase in focal lesions and in zone I of rat liver after treatment with chemical carcinogens

-Glutamyltranspeptidase (-GT) is known to be increased in putativepre-neoplastic foci but also in the periportal zone I of ratliver under a variety of circumstances not directly relatedto carcinogenesis. To be able to distinguish between these twoinstances -GT was studied by enzyme determination in micro-dissectionsobtained from the two locations and by both histochemical andimmunohistochemical staining in serial sections. Altered hepaticfoci and alterations in zone I were produced in three modelsof hepatocarcinogenesis: (i) initiation by N-nitrosomorpholineand tumor promotion by phenobarbital, (ii) continuous administrationof 2-acetyl-aminofluorene and (iii) continuous administrationof meta-pyrikne hydrochloride. In micro-dissections-GT activitywas similarly increased in focal lesions and in zone I afterfeeding methapyrilene. Histochemically detectable -GT, stainedaccording to Rutenburg et al. (23), was observed both in zoneI and in focal lesions. Focal lesions were also ATPase negativeand UDP-glucuronyhransferase positive in all three models. -GTin focal lesions could be selectively detected by immunohistochemicalstaining using antibodies to the rat kidney enzyme and an indirectperoxidase reaction. These findings suggest immunochemical differencesbetween -GT in focal lesions and in zone I.     

Comparative histochemical investigation of the glutathione S-transferase placental form and {gamma}-glutamyltranspeptidase during N-nitrosobis(2-hydroxypropyl)amine-induced pancreatic carcinogenesis in hamsters

Immunohistochemical staining using anti-rat glutathione S-transferaseplacental form (GST-P) rabbit antibody and enzyme histochemicalstaining for -glutamyltranspeptidase (-GT) were investigatedin putative preneoplastic lesions and adenocarcinomas in thepancreas of Syrian golden hamsters treated with N-nitrosobis(2-hydroxypropyl)amine(BHP). Areas with ductular proliferation, ductal hyperplasia,and intraductal carcinoma were strongly positive for GST-P bindingand negative for -GT. Cystic adenoma, microcarcinoma, and carcinomaswere constantly positively stained by GST-P and partially positivefor -GT. GST-P appears to be useful as a positive marker forputative preneoplastic lesions in pancreatic carcinogenesis.Since normal acinar cells are strongly positive for -GT, thefindings might suggest that acinar cells constribute to thedevelopment of cystic adenoma, microcar-cinoma, and carcinomas.     

Utilization of 1, N6-Etheno-2'-deoxyadenosine 5'-triphosphate during DNA synthesis on natural templates, catalyzed by DNA polymerase I of Escherichia coli

To test whether vinyl chloride-induced mutagenesis might involveambiguous base pairing of 1, N⁶-etheno-adenine (A) during DNAsynthesis, we examined the base pairing potential of dATP duringDNA synthesis catalyzed by Escherichia coli DNA polymerase I(Klenow fragment). An electrophoretic assay of chain elongationwas used to assess the degree to which dATP could substitutefor each of the normal dNTPs during elongation of a primer annealedto a bacteriophage template. Despite the fact that the ethenobridge completely blocks normal Watson-Crick pairing of A withT, we observed that dATP could substitute for dATP during primerelongation (although inefficiently). In addition, detectablesubstitution of dATP for dGTP and dCTP occurred, indicatingthat A exhibits ambiguous base pairing properties. The relativeease of dAMP incorporation (opposite template T, C and G) appearedto vary considerably at different positions along the template.The major, form of eA incorporation (replacement of A) was confirmedby measurements of dATP-dAMP turnover (a commonly used methodfor detecting misincorporation), and also by the demonstrationthat A was present in enzymatic hydrolysates prepared from DNAthat was synthesized with dATP replacing dATP. A model for ambiguousbase pairing of dATP is proposed, in which incorporation occursvia the protonated, syn form of dATP.     

Differential regulation of two microsomal epoxide hydrolases in hyperplastic nodules from rat liver

Two microsomal epoxide hydrolases of the rat liver were foundto be differentially regulated in hyperplastic nodules. Whilstthe activity for substrates of the well-known microsomal epoxidehydrolase with a broad substrate specificity (EHb), benzo[a]pyrene4,5-oxide and androstene oxide (16,17-epoxyandrosten-3-one),was greatly (5-fold) increased in the nodule microsomes andmoderately (2-fold) increased in the surrounding tissue, thatfor the substrate of the novel microsomal epoxide hydrolase,cholesterol 5, 6-oxide (EH_ch) remained unchanged. Since bothenzymes convert endogenous steroid epoxides but with distinctstructural features, this differential regulation may indicatea role of endogenous steroid epoxide(s) of a defined structureduring hepatocarcinogenesis. Alternatively, this differentialregulation may serve as a marker during hepatocarcinogenesis.     

TGF-{alpha} and EGF-receptor mRNAs in human oral cancer

Transforming growth factor alpha (TGF-) and epidermal growthfactor receptor (EGFR) have been shown to be present in mostsquamous cell carcinomas. Using the Syrian hamster oral cancermodel, we have recently demonstrated the consistent presenceof TGF- and EGFR mRNAs in chemically transformed hamster oralkeratmocytes. We now present evidence that in human oral cancer(in vivo and in vitro), TGF- and EGFR mRNAs can also be consistentlydetected. No TGF- mRNA can be detected in normal human oralepithelium by Northern blot analysis. These findings reinforcethe use of the hamster cheek pouch as an experimental modelfor the study of oral cancer development, at least in referenceto the possible participation of TGF- in the malignant transformationprocess.     

Uptake of taurocholate by isolated {gamma}-glutamyltranspeptidase positive, putatively preneoplastic hepatocytes from 2-acetylaminofluorene treated rats

Uptake of the bile acid taurocholate was determined in livercells isolated from male Wistar rats fed a standard diet ora diet containing 2-acetylaminofluorene (2-AAF). In addition,uptake was analysed in unaltered, -glutamyltranspeptidase (-GTase)negative, and in putatively preneoplastic, -GTase positive,hepatocytes separated from the total liver cell preparationisolated from the 2-AAF-treated animals. Total hepatocytes fromthe carcinogen-treated rats showed a - 50%decrease in the maximumrate of taurocholate transport compared to cells from untreatedanimals. Bile acid uptake in -GTase positive and negative livercells revealed that V_max was decreased by 44 ± 14% inthe preneoplastic cell population. Since the K_m value did notdiffer significantly it appears that the number of carrier moleculesis reduced in the early preneoplastic hepatocytes. Our resultsshow a partial loss of a liver-specific function in early preneoplastic-GTase positive hepatocytes.     

UVR induction of TGF{alpha}: a possible autocrine mechanism for the epidermal melanocytic response and for promotion of epidermal carcinogenesis

The occurrence of the epidermal growth factor homologue, transforminggrowth factor (TGF), in embryonic and neoplastic tissues suggeststhat it may he an oncofetal version of epidermal growth factor.A strong case is developing for TGF to have an autocrine modeof action in sustaining the autonomous growth of several typesof tumour. We propose that TGF normally has an autocrine rolenot only in stimulating the growth of some fetal tissues butalso with postnatal epidermal cells in response to local stimuli—inparticular ultraviolet radiation (UVR). As a first step to testthis hypothesis we have checked whether UVR will induce theproduction of TGF, measured by radioimmunoassay, using a highlyspecific monodonal antibody which recognizes native, biologicallyactive human TGF. We found that cultures of normal foreskinmelanocytes do not produce detectable amounts of TGF when grownunder routine conditions, but, within 12 h of exposure to lowdoses of short-wavelength UVR, significant quantities of TGFare produced. The UVR-induced TGF is both cell associated andreleased into the medium of these cultures. Also, UVR has apromoting action on epidermal cells which have been initiatedby carcinogenic activity. A significant part of the promotingactivity may be due to autocrine stimulation of multiplicationof partially transformed epidermal cells. In this regard wefound that UVR induced TGF in HeLa cells and all human melanomalines so far tested. Induction was complete within 24 h of asingle exposure. Dose-response curves of TGF induction in amalignant melanoma cell line showed a distinctive peak of factorinduced by low (2 J/m²) doses of UVR. Higher doses which inhibit[³H]thymidine incorporation resulted in lower levels of inducedTGF. These findings are consistent with the participation ofTGF as an autocrine mediator of UVR-induced tumour promotion,as well as cell multiplication, in sun-exposed skin.     

Expression of glutathione S-transferase isoenzymes in human small cell lung cancer cell lines

A glutathione S-transferase (GST) isoenzyme having common antigenicityto rat placental form (GST-P) and human placental form (GST-)has recently been suggested may be a marker of carcinogenesis.In the present study we have investigated the expression ofthis isoenzyme in three small cell lung cancer cell lines inorder to determine whether or not this isoenzyme can be usedas a general marker of carcinogenesis. GST activity towards1-chloro-2,4-dinitrobenzene in two of the cell lines (NES andNOC-361) was moderately higher than that in normal human lung,but this activity was markedly suppressed in one of the celllines (NCI-H69). Quantitation of the GST isoenzymes in the tumorsgrown in nude mice by injecting these cell lines also revealeda moderate increase of GST--type isoenzyme in NES and NOC-361and its suppression in NCI-H69. Immunocytochemical localizationstudies with these tumors using antibodies raised against GST-also indicated a drastic decrease of GST--type isoenzyme inNCI-H69 and this finding was confirmed by Western biot studies.These results suggest that GST- or the isoenzyme(s) having similarimmunological nature to GST-, cannot be used as the generalmarkers of neoplastic states.     

Inhibitory effect of {alpha}-tocopherol on the formation of nitrosomorpholine in mice treated with morpholine and exposed to nitrogen dioxide

The possibility that -tocopherol (vitamin E) inhibits the formationof nitrosomorpholine (NMOR) in vivo was investigated in miceorally pretreated with -tocopherol (2.5–100 mg/kg bodywt) once daily for 6 days. Twenty-four hours later, the animalswere injected i.p. with 2 mg of morpholine (MOR) per animalfollowed by exposure txo 47 p.p.m. of NO₂ for 2 h. Under theseconditions, low oral doses of -tocopherol (2.5–5 mg/kgbody wt) significantly decreased NMOR formation in vivo. Astotal body -tocopherol levels increased, in vivo NMOR formationdecreased, and a maximal 50–70% inhibition of NMOR formationoccurred at -tocopherol levels of 5 µg/g body wt. Additionalresults showed that NMOR was rapidly eliminated in mice, sothat studies which measure the levels of NMOR found in animalstreated with MOR and then exposed to NO₂ may underestimate theamount of NMOR that is actually formed. Finally, oral pretreatmentof up to 100 mg of -tocopherol/kg body wt had no effect on NMORelimination.