Effects of selenium on 1,2-dimethylhydrazine metabolism and DNA alkylation

Sodium selenite (Se) decreases the incidence of colon tumorsinduced in rats by 1,2-dimethylhydrazine (DMH). In order todetermine the basis for this inhibition, we studied the effectsof Se on DMH metabolism, DNA alkylation and the rate of cellturnover of the target tissue. The effects of Se pretreatment(4 p.p.m. in the drinking water, for 2, 4 or 6 weeks) on DMHmetabolism were monitored in male Sprague-Dawley rats by measuringexpired ¹⁴CO₂ and azo[¹⁴C]methane over a 12 h period after as.c. injection of [¹⁴C]DMH (20 mg/kg body weight). Comparedto control rats, which received only [¹⁴C]DMH, Se pretreatmentcaused an increase in exhaled azomethane (31–69%) anda corresponding decrease in ¹⁴CO₂ (4–33%) as the lengthof treatment increased from 2 to 6 weeks. The extent of DNAalkylation (measured as N-7 and O⁶-methylguanine formation)after Se pretreatment was reduced 20–27% in liver andwas increased 40–43% in colon. Metabolic incorporationof [¹⁴C] from [¹⁴C]DMH into adenine and guanine (presumablyvia Q pathways) was reduced 69–72% in colon DNA of Se-treatedrats and [3H]thymidine incorporation was reduced 61 –65%. This may have been due to decreased cell turnover. A similarresponse was not observed in the liver. The data suggest thatSe decreases hepatic DMH metabolism, and that this may be compensatedby an increase in ex-trahepatic metabolism and alkylation. Althoughcolon alkylation is increased by Se pretreatment, fewer tumorsresult. This may be due to a decrease in DNA synthesis in thistissue.     

Uptake and metabolism of {beta}-carotene and retinal by C3H/10T1/2 cells

Both ß-carotene (ß-C), a vitamin A precursor,and vitamin A itself have been shown to reversibly inhibit neoplastictransformation in 10T1/2 cells during the progression phaseof carcinogenesis. In order to determine whether the activityof ß-C in these cells may be attributed to conversionto vitamin A or is intrinsic to the carotenoid molecule, theuptake and metabolism of ß-C, and of retinal, theimmediate product of dioxygenase-cleavage of ß-C,was studied in 10T1/2 cells. Cellular uptake of 2.6 nmol/10⁶cells occurred 24h after treatment with 10^–5 M ß-C.Thereafter, cell levels remained relatively stable between 1and 2 nmol/10⁶ cells over the 1-week treatment period. Uponremoval of ß-C from the medium, cellular levels decreasedby {small tilde}80% in 2 weeks, then stabilized. Retinal wasrapidly and quantitatively converted to retinol by 10T1/2 cells,suggesting that the inhibitory action of retinal on neoplastictransformation in these cells is due to its conversion to retinol,and that any enzymatic conversion of ß-C to retinalby these cells would be expected to result in retinol as theend product. Using [¹⁴C]ß-C, we found no evidencefor formation of [¹⁴C]retinol, [¹⁴C]retinal or [¹⁴C]retinoicacid using sensitive HPLC. We therefore conclude that ß-Chas intrinsic chemopreventive activity in 10T1/2 cells, perhapsdue to its anti-oxidant properties.     

Supplemental dietary calcium fails to alter the acute effects of 1,2-dimethylhydrazine on O6-methylguanine, O6-alkylguanine-DNA alkyltransferase and cellular proliferation in the rat colon

Prior studies from our laboratory have demonstrated that K-rasG to A mutations were detectable in a high percentage of carcinomaswhich developed in the colons of animals treated with the knowncolonic procarcinogen, 1,2-dimethyl-hydrazine (DMH). Moreover,in this model, the incidence of these mutations was decreasedby a supplemental dietary calcium regimen which concomitantlydecreased the frequency of rats with multiple tumors as wellas tumor size. In an attempt to clarify the possible mechanism(s)involved in this antimutagenic effect of supplemental calcium,two groups of Sprague—Dawley rats were fed semisyntheticdiets containing either 0.87 or 1.80% cakium by weight for 3weeks, s.c. injected with 100 mg/kg of DMH and killed priorto and at various time periods (16-144 h) after injection. Thecolons of animals were analyzed and compared with respect toO⁶-methylguanine content in DNA, O⁶-alkylguanine-DNA alkyltransferaselevels as well as cellular proliferation, as assessed by immunohistochemicalstaining of colonic crypts by bromodeoxyundine. In certain experiments,these parameters were also analyzed in the proximal and distalcolon before and at various times after administration of DMH.The results of these experiments demonstrated that supplementaldietary calcium was not found to influence significantly O⁶-methylguaninelevels, alkyltransferase levels or cellular proliferation inthe entire colon or in either colonic segment before or afterthe acute administration of DMH. DMH did, however, differentiallyalter all three of these biochemical parameters in the colonicsegments (distal > proximal), possibly due to a greater degreeof metabolic activation in the distal colon.     

Inhibition by phenylethyl and phenylhexyl isothiocyanate of metabolism of and DNA methylation by N-nitrosomethylamylamine in rats

We investigated the effect of 2-phenylethyl and 6-phenylhexylisothiocyanate (PEITC and PHITC) on the metabolism of the ratesophageal carcinogen, N-nitrosomethylamylamine (NMAA). PEITCwas administered orally to MRC-Wistar rats as single doses of0.1 or 1.0 mmol/kg, or by other regimens. When esophagi andliver slices from the treated rats were incubated with 23 µMNMAA, the formation of 2-to 5-hydroxy-NMAA was inhibited by45–90% for esophagus and by 14–19% for liver slices.In contrast, when esophagi and liver slices from untreated MRC-Wistarrats were incubated in vitro with NMAA and 10 µM PEITC,the PEITC inhibited hydroxy-NMAA formation similarly (by 79–89%)in the two tissues. Also, PEITC inhibited the formation fromNMAA of the hydroxy-NMAAs, formaldehyde and pentaldehyde byesophageal and liver microsomes to similar extents. In studieson DNA methylation by NMAA, 7- and O⁶-methylguanine (O⁶-MeG)were determined by HPLC with fluorimetric detection. Guaninemethylation in esophageal and liver DNA was generally closeto linear for doses of 5–50 mg NMAA/kg. With 50 mg NMAA/kg,guanine methylation in esophageal and liver DNA peaked after5 h, and 8–11% of the peak O⁶-MeG persisted after 72 h.A single dose of 0.1 or 1.0 mmol PEITC/kg reduced the O⁶-MeGlevels by 44–51% in the esophagus but by only 7–22%in the liver. Administration of the PEITC homolog, PHITC, inhibitedNMAA metabolism by liver slices from the treated rats and themethylation of guanine in liver DNA, but had little effect inthe esophagus, i.e. PHITC tended to have the opposite tissuespecificity to PEITC. The finding that administration of PEITCspecifically inhibited NMAA metabolism in the rat esophagussupports the view that PEITC may be a useful chemopreventiveagent against esophageal carcinogenesis in humans.     

Extent of DNA 2-hydroxyethylation by N-nitrosomethylethylamine and N-nitrosodiethylamine in vivo

At low doses, N-nitrosomethylethylamine (NMEA) selectively producesliver tumors in rats, whereas ß-trideuterated NMEAalso includes esophageal carcinomas under these conditions.Since deuteration is capable of retarding enzymic hydroxylation,these studies suggest that ß-hydroxylation plays asignificant role in the organ specificity of NMEA. To test thehypothesis that this metabolic pathway occurs in vivo to yielda hydroxyethylating intermediate, we have determined the extentof hydroxyethylation of hepatic DNA in male Fischer 344 ratsfollowing a single i.p. injection of [1-ethyl-¹⁴C]NMEA (6.3mg/kg, 4 h survival). After hydrolysis in 0.1 M HCI, DNA purineswere analysed by cation exchange chromatography. Of the majoralkylpurines identified, 7-ethylguanine (7-etG) (6.7 µmol/molguanine) and O⁶-ethylguanine (4.1 µmol/mol guanine) comprised13 and 8% of the eluted radioactivity, respectively. 7-(2-HydroxyethyI)guanine(7-heG) was the only hydroxyethyl adduct detectable, and comprisedless than 2% of the amount of 7-etG. 3-Ethylguanine and 3- and7-ethyladenine were also identified as products of NMEA metabolism.Similar analyses were carried out on hepatic DNA from rats treatedwith N-nitrosodi[1-¹⁴C]ethylamine (6.9 mg/kg, 4 h survival).Only trace amounts of 7-heG could be detected. The very lowconcentrations of ß-hydroxyethylated DNA bases observedsuggest that this route of metabolism does not contribute significantlyto the carcinogenicity of these compounds.     

Marked increase in urinary excretion of nitrate and N-nitrosothioproline in the osteogenic disordered syndrome rats, lacking ascorbic acid biosynthesis, by administration of lipopolysaccharide and thioproline

Urinary excretions of nitrate and N-nitrosothiazolidine-4-carboxylicacid (N-nitrosothioproline; NTPRO) were determined in rats withosteogenic disordered syndrome (ODS, od/od), lacking L-ascorbicacid (ASC) biosynthesis, after i.p. administration of Escherichiacoli lipopolysaccharide (LPS, 1 mg/kg) followed by thiazolidine-4-carboxylicacid (thioproline, 20 mg/rat). L-Ascorbic acid-sufficient ODSrats showed the excretion of nitrate and NTPRO at the levelsof 203 ± 7.9 µmol/24 h and 369 ± 111 pmol/24h respectively, whereas the levels of nitrate and NTPRO in ASC-deficient(scorbutic) rats increased to 54.7 ± 5.6 µmol/24h (P < 0.01) and 796 ± 367 pmol/24 h (P < 0.05)respectively. Administration of L-arginine further increasedurinary excretion of nitrate and NTPRO while D-arginine showedno effect. N^G-Monomethyl-L-arginine, a specific inhibitor ofnitric oxide synthase (NOS), strongly inhibited endogenous formationof both nitrate and NTPRO. These results indicate that increasedexcretion of NTPRO in ODS rats stimulated by LPS involves inductionof NOS leading to an increase in endogenous formation of reactivenitrogen oxides such as N₂O₃, a potent nitrosating agent atphysiological pH conditions. Increased NOS activities in theplasma and various tissues of ODS rats were observed 5 h aftertreatment with LPS. The possibility of extragastric N-nitrosocompound formation in inflammation sites is discussed.     

Formation of a glucuronide conjugate of 12-O-tetradecanoylphorbol-13-acetate by LLC-PK1 renal epithelial cells in culture

The metabolism of [³H]tetradecanoylphorbol-13-acetate [³H]TPA)was studied in LLC-PK₁ cells, a differentiated renal epithelialcell line. In contrast to earlier studies in both fibroblasticand epithelial cells, a major metabolite of [³H]-TPA by thesecells was a new, previously unobserved species which was muchmore hydrophilic than TPA or its major metabolite in other cells,phorbol-13-acetate. Incubation of culture medium containingthis metabolite with ß-glucuronidase greatly reducedthe amount of this polar metabolite, accompanied by a nearlyequal increase in the amount of [³H]-TPA extractable by organicsolvents from this medium. In several experiments this TPA-glucuronicacid conjugate accounted for 20–50% of the total metabolitespresent after several days of incubation of LLC-PK1 cells with[³H]TPA. Two other polar renal epithelial cell lines, derivedfrom canine (MDCK) and bovine (MDBK) kidney, were incapableof converting [³H]TPA to a ß-glucuronidase sensitiveproduct. A time course study indicated that once formed, theTPA-glucuronic acid conjugate was stable in LLC-PK₁ cells. Incubationof LLC-PK₁ conditioned medium containing the conjugate withother cell types such as primary hamster embryo cells or primarynewborn hamster epidermal cells indicated that this materialwas stable in these cultures also. However, primary culturesof rat hepatocytes, and to a lesser extent a human hepatomacell line, were capable of further metabolism of this conjugateto a form insensitive to ß-glucuronidase treatment.This is the first evidence for the formation of a TPA-glucuronicacid conjugate either in vivo or in vitro, and while it is stablein the presence of the cells which produce it, this compoundcan also be further metabolized, presumably via ß-glucuronidasein at least one cell type (i.e., hepatocytes). The biologicalsignificance of the formation and cleavage of this TPA-glucuronicacid conjugate remains to be determined.     

Distribution of the carcinogenic tryptophan pyrolysis product Trp-P-1 in control, 9-hydroxyellipticine and {beta}-naphthoflavone pretreated mice

Autoradiograms obtained 1–4 h after i.v. injection ofthe ¹⁴C-labelled carcinogenic tryptophan pyrolysis product Trp-P-1to albino and pigmented mice showed a pronounced uptake of radioactivityin the lymphatic system (thymus, lymphnodes, bone marrow andspleen), in the endocrine system (hypophysis, thyroid, adrenalmedulla) and in the liver, kidney medulla and brain. High radioactivitywas present in the excretory pathways, predominantly in thebile/intestinal contents. At longer post-injection times (24h to 6 days) most of the labelled substance had left the tissues,except for the liver which still retained a high concentrationof radioactivity. Trp-P-1 is known to be activated by cytochromeP-448. The uptake of radioactivity in the liver could be reducedby pretreatment with the cylochrome P448 inhibitor 9-hydroxy-ellipticinesuggesting that the observed accumulation of radioactivity inthe liver was partly due to metabolites of Trp-P-1. After pretreatmentwith the cytochrome P-448 inducer ß-naphthoflavone,the administration of Trp-P-1 resulted in a highly selectiveaccumulation of radioactivity in the lung parenchyma, exceedingall other tissues in the body. ß-Naphthoflavone pretreatmentalso increased the uptake of radioactivity in the kidney cortexand small intestinal mucosa. As indicated by a high labellingof the pigmented tissues of the maternal and fetal eye, thecarcinogen and/or its metabolites were accumulated in melanin.     

Potentiation of alkylation-induced sister chromatid exchange frequency by 3-aminobenzamide is mediated by intracellular loss of NAD+

3-Aminobenzamide (3AB) is a competitive inhibitor of poly-(ADP-ribose)polymerase. It will interact synergistically with certain monofunctionalalkylating agents to increase the frequency of sister chromatidexchanges (SCEs) in Chinese hamster ovary (CHO) cells. 3AB willalso increase the baseline SCE frequency in exposed cells. Theextent of interaction between 3AB and monofunctional alkylatingagents varies depending on the alkylating agent used and appearsto be due to the different amounts of membrane damage producedby the alkylating agents. In this study, exogenously added ß-NAD⁺was found to reduce substantially SCE frequency in cells thathad been treated with combinations of 3AB and methyl methanesulfonate(MMS) but not in cells treated with 3AB and N-methyl-N'-nitro-N-nitrosoguanidine(MNNG). MMS produces more cell membrane damage than MNNG atequitoxic doses. ß-NAD⁺ is the substrate for ADP-ribosylationand normally does not freely diffuse into cells. ß-NAD⁺had no significant effect on SCE induction in intact cells orin cells treated with either 3AB or alkylating agent alone.In contrast to ß-NAD⁺, exogenously added -NAD⁺, whichis an inhibitor of poly(ADP-ribose) polymerase, increased SCEfrequency in MMS-treated cells. Thus the interaction between3AB and certain monofunctional alkylating agents in SCE formationis apparently due to cell membrane permeabilization and theloss of intracellular NAD⁺ which in turn probably results ina greater inhibition of ADP-ribosylation in the presence of3AB.     

Induction of a DNA adduct detectable by 32P-postlabeling in the dorsolateral prostate of NBL/Cr rats treated with estradiol-17{beta} and testosterone

Treatment with estradiol-17ß and testosterone inducesepithelial dysplasia and, subsequently, adenocarcinoma in thedorsolateral prostate of NBL rats. The purpose of this studywas to determine whether this carcinogenic effect is mediatedby genotoxicity. Analogous to adducts produced by estrogensin the male hamster kidney, a target of estrogen carcinogenicity,induction of DNA adducts detectable by ³²P-postlabeling wasinvestigated in the prostate target tissue. NBL rats were treatedwith separate Silastic tubing implants containing testosteroneand estradiol-17ß. Control animals received emptyimplants. Animals were killed at 8, 16 and 24 weeks after initiationof treatment, and accessory sex glands were sampled for adductanalysis. DNA of the dorsolateral and ventral prostate and thecoagulating gland (= anterior prostate) was isolated and analyzedby nuclease Pl-enhancement of the ³²P-postlabeling assay. DNAadducts were quantitated by Cerenkov counting. An adduct occurredselectively in DNA of the dorsolateral prostate of rats treatedwith estradiol plus testosterone for 16 or 24 weeks with relativeadduct level values of     

Topical treatment of mice with benzo[a]pyrene or parenteral administration of benzo[a]pyrene diol epoxide--DNA to rats results in faecal excretion of a putative benzo[a]pyrene diol epoxide--deoxyguanosine adduct

The administration of [³H]BPDE-DNA, whether by i.p. or i.v.injection, to male Wistar rats resulted in the majority of theradioactivity being recovered in the faeces. Excretion was rapid:within 24 h post-injection, 45% of the applied dose was recoveredin the faeces. H.p.l.c. analysis of radioactive material extractedfrom the faeces by methanol showed that it contained a singlecomponent which co-chromatographed with [³H]BPDE-dGuo and whichwas not affected by treatment with alkaline phosphatase, arylsulphatase or ß-glucuronidase. To determine if thisphenomenon occurs after topical application of BP to a targettissue, such as mouse skin, animals were treated with [³H]BPand their faeces collected. After an extensive extraction procedureinvolving differential solubility in organic solvents, SephadexLH-20 chromatography and h.p.l.c, a product was isolated frommice faeces which had characteristics consistent with a [³H]BPDE-dGuoadduct. These findings are discussed in relation to detectionof BPDE adducts in human populations.     

Metabolism of the carcinogenic air pollutant 2-nitrofluorene in the rat

After a single oral dose of 2-nitro [9-¹⁴C]fluorene to rats,radioactivity with associated mutagenic activity was rapidlyexcreted in both urine and faeces, urine being the major excretoryroute. The major part of the urinary mutagenicity was associatedwith the unconjugated fraction of metabolites which consistedof a range of hydroxylated acetylaminofluorenes in additionto a number of hydroxylated nitrofluorenes. The formation ofhydroxylated acetylaminofluorenes supports the contention that2-nitrofluorene, in vivo, enters the metabolic pathway of thewell-known carcinogen 2-acetylaminofluorene. The single mostmutagenic metabolite of 2-nitrofluorene was a hydroxylated nitrofluorene;the formation of this metabolite was markedly increased whenthe rats had been treated with ß-naphthoflavone. Finally,an alternative pathway is discussed for the formation of 1-and 3-hydroxyacetylaminofluorenes.     

{beta}-Carotene and canthaxanthin inhibit chemically- and physically- induced neoplastic transformation in 10T1/2 cells

We have studied the effects of ß-carotene(ß03-C),a vitamin A precursor of plant origin, and canthaxanthin (CTX),a non-provitamin A carotenoid, on the neoplastic transformationof C3H/10T1/2 murine fibroblast cells. Chemical transformationin this well-characterized cell system has previously been shownto be reversibly inhibited by retinoids, compounds with vitaminA-like activity. Here we show that both ß-C and CTXinhibit 3-methycholanthrene (MCA)-induced transformation withED⁵⁰S of 9 x10-⁷M and 2x10-⁷ M, respectively. Both carotenoidsfailed to inhibit X-ray-induced transformation when the cellswere treated prior to and during irradiation. However, whenthe drugs were added 1 week after X-irradiation and maintainedin the medium thereafter, as in the chemical transformationprotocol, both carotenoids inhibited subsequent developmentof transformed foci in a dose-dependent manner. Again, CTX wasmore effective than ß3-C, such that 3 x 10-⁶M completelyinhibited radio-genicaly-induced foci. Similar to the previouslydescribed action of retinoids, the inhibition of MCA-inducedtransformation was reversible; developed upon removal of thedrug, transformed foci developed within 2 weeks, indicatingthat the carotenoids were not specifically toxic to initiatedcells. Although both carotenoids caused a small dose-dependentdecrease in the growth rate of both parental and initiated 10T1/2cells, they did not markedly affect colony size or number whenthe cells were treated as in the transformation assays, nordid they influence the expression of neoplasia of two transformedcell lines. Although the actions of ß3-C and CTX aresimilar to those of retinoids in the 10T1/2 system, we suggestthat the carotenoids act via a different mechanism, since CTXcannot be converted to active retinoids in mammalian cells,and there is no evidence that 10T1/2 cells can convert ß-Cto vitamin A. We suggest that the carotenoids, lipid anti-oxidantproperties may be responsible for their inhibitory actions ontransformation.     

Detection of dopamine DNA adducts: potential role in Parkinson's disease

Oxidation of catecholamines may lead to the formation of O-semiquinonesand o-quinones in catecholaminergic brain tissues, and thesereactive molecules may form DNA or protein adducts. In thisstudy, cultured cells were treated with dopamine (DA) for ²⁴h and ³²P-postlabeling was used to detect DA-DNA adducts. InHL–60 cells, 250µM DA induced 8.5 DNA adducts/108nucleotides; adduct formation was dose-dependent up to 500 µMDA. Addition of H2O2 increased the relative adduct levels ⁷-to 13-fold, but no adducts were detected when DA and ascorbicacid were added simultaneously. In human glioblastoma cell linesU87, U251, SF-763 and SF-767, 1000 µM DA produced 0.98–2.31adducts/108 nucleotides. These results suggest that the formationof DNA adducts by DA may contribute to the development of certainneurodegenerative diseases such as Parkinson's disease.     

Characterization and properties of the DNA adducts formed from N-methyl-4-aminoazobenzene in rats during a carcinogenic treatment regimen

Chronic oral administration of the carcinogenic aminoazo dyeN-methyl-4-aminoazobenzene (MAB) to rats is known to resultin the induction of liver tumors. In order to assess the roleof carcinogen-DNA adduct formation in MAB hepatocarcinogenesis,male rats were fed 0.06% [3'-³H]MAB in the diet for 1, 3 or5 weeks. Groups were sacrificed at 0, 24 and 72 h after dosing,and DNA was isolated from the liver and from two non-targettissues, the kidney and spleen. Upon enzymatic hydrolysis ofthe DNA, [³H]aminoazo dye-nucleoside adduct levels in thesetissues were determined by h.p.l.c. Rats concurrently administeredunlabeled MAB for 5 weeks and continued on a control diet for9 months developed hepatocellular carcinomas (16/30 animals).No tumors were observed in 21 rats given only control diets.After chronic administration of [³H]MAB, three major MAB-DNAadducts were found in vivo: N-(deoxyguanosin-8-yl)-MAB (C8-dG-MAB),3-(deoxyguanosin-N²-yl)-MAB (N²-dG-MAB) and 3-(deoxyadenosin-N⁶-yl)-MAB(N⁶-dA-MAB). In addition, several minor products were identifiedas: (i) an (8,9)-purine ring-opened derivative of C8-dG-MABthat may represent an intermediate in DNA repair; (ii) N-guanosin-8-yl-MABwhich is present due to trace RNA contamination; (iii) cis isomersof C8-dG-MAB and N-guanosin-8-yl-MAB, formed by photo-illuminationduring analyses; and (iv) N-(guanin-8-yl)-MAB, a deribosylatedproduct resulting from thermal depurination of C8-dG-MAB. Inaddition, N-(deoxyguanosin-8-yl)-4-aminoazobenzene (C8-dG-AB),a major adduct previously detected in mouse liver after a singledose of 4-aminoazobenzene, was found in rat liver but appearedto be present in significant amounts only after chronic treatmentwith MAB. This product co-chromatographed with N⁶-dA-MAB butcould be removed by selective decomposition in 0.1 N NaOH. Forall tissues examined N²-dG-MAB and C8-dG-MAB were the majoradducts observed with each accounting for 40-50% of the totalcarcinogen bound to DNA in rats that were sacrificed immediatelyafter MAB feeding for 1, 3 or 5 weeks. The levels of total MAB-DNAadducts in the liver were 2–10 times greater than in thekidney or spleen and appeared to increase 2- to 3-fold overthe dosing period. However, by 24–72 h after cessationof MAB treatment, hepatic C8-dG-MAB showed a rapid decline tolevels similar to that found in non-target tissues. The minoradducts, N⁶-dA-MAB and C8-dG-AB, exhibited similar behaviorand never accounted for > 5–10% of the total DNA binding.In contrast, hepatic N²-dG-MAB was a persistent lesion throughoutthe treatment regimen; at 72 h after dosing, it accounted for60–90% of the hepatic DNA adducts and was the only adductwhose levels correlated with target tissue specificity aftera complete hepatocarcinogenic dose of MAB.     

Loss of p53 enhances the induction of colitis-associated neoplasia by dextran sulfate sodium

Loss of p53 function is an early event in colitis-associatedneoplasia in humans. We assessed the role of p53 in a mousemodel of colitis-associated neoplasia. Colitis was induced inp53^–/–, p53^+/– and p53^+/+ mice using threeor four cycles of dextran sulfate sodium (DSS) followed by 120days of water. Mice were examined for incidence, multiplicityand types of neoplastic lesions. Lesions were examined for mutationsin ß-catenin (exon 3), K-ras (codons 12/13) and p53(exons 5–8) by sequencing and for cellular localizationof ß-catenin by immunohistochemistry. The incidenceof neoplastic lesions was 57, 20 and 20% in p53^–/–,p53^+/– and p53^+/+ mice, respectively (P = 0.013). p53^–/–mice had a greater number of total lesions (P < 0.0001),cancers (P = 0.001) and dysplasias (P = 0.009) per mouse thaneither p53^+/– or p53^+/+ mice. Flat lesions were associatedwith the p53^–/– genotype, whereas polypoid lesionswere associated with the p53^+/– and p53^+/+ genotypes (P< 0.0001). ß-Catenin mutations were present in75% of lesions of p53^+/+ mice and absent in lesions from p53^–/–mice (P = 0.055). Nuclear expression of ß-cateninwas seen only in polypoid lesions (91%). No K-ras or p53 mutationswere detected. These data indicate that loss of p53 enhancesthe induction of colitis-associated neoplasia, particularlyflat lesions, and dysregulation of ß-catenin signalingplays an important role in the formation of polypoid lesionsin this mouse model. As observed in humans, p53 plays a protectiverole in colitis-associated neoplasia in the DSS model. Abbreviations: CRC, colorectal cancer; DSS, dextran sulfate sodium; PCR, polymerase chain reaction; UC, ulcerative colitis; PGE₂, prostaglandin E₂ Received January 24, 2007; revised May 11, 2007; accepted May 29, 2007.     

Metabolism of the food carcinogen 2-amino-3, 8-dimethylimidazo[4, 5-f]quinoxaline in isolated rat liver cells

2-Amino-3, 8-dimethylimidazo[4, 5-f]quinoxaline (MelQx) wastransformed to at least 10 metabolites in suspensions of hepatocytesisolated from Aroclor 1254 treated rats. Combining biochemicaldata such as effects on MeIQx metabolism of metabolic modulatorsand incorporation of radioisotopic sulfur with UV, mass and¹H-NMR spectroscopy, we elucidated the structures of six metabolites.About 40% of the MeIQx was transformed to 2-amino-3, 8-dimethylimidazo[4,5-f]quinoxalin-4(or5)-yl sulfate. Other oxygenated metaboliteswere 2-amino-8-hydroxymethy1–3-methylimidazo[4, 5-f)quinoxalin-4(or5)-yl sulfate and 2-amino-4(or5)-ß-D-glucuronopyranosyloxy-3,8-dimethylimidazo[4, 5-f]quinoxaline. Evidence was obtainedthat a glutathione conjugate was formed. This metabolite, andthe other oxygenated metabolites were probably formed in P-450catalyzed reactions. Two metabolites, 2-ß-D-glucurono-pyranosylamino-3,8-dimethylimidazo[4, 5-f)quinoxaline and the N(3, 8-dimethylimidazo[4,5-f]quinoxaline-2-yl)sulfamate, were direct conjugates of MeIQx.     

H.p.l.c. of benzo[a]pyrene glucuronide, sulfate and glutathione conjugates and water-soluble metabolites from hamster embryo fibroblasts

Chromatography of benzo[a]pyrene (BaP) sulfate, glucuronideand glutathione (GSH) conjugate standards were examined by h.p.l.c.on a C8 column as modified by various organic acids and solvents.Sulfate and glucuronide standards were positional isomers derivedfrom BaP-1,3,6,7,9 phenols and BaP-GSH conjugates consistedof a racemic mixture of BaP-4,5-GSH. In the absence of acid,BaP conjugates appeared as rapidly eluting, unresolved peaksin aqueous-methanol or acetonitrile gradients or coeluted asbroad peaks in a water-propanol gradient, with the exceptionof BaP-7-OH sulfate which eluted as a distinct symmetrical peak.Addition of acetic or trifluoroacetic (TFA) acids enhanced columnretention of BaP conjugates in each solvent system. Upon acidificationof mobile phases, BaP-GSH isomers were partially resolved, isomersof BaP sulfates or of BaP glucuronides coeluted, and BaP-7-OHsulfate was resolved from all conjugates. BaP-GSH conjugateswere most resolved and preceded elution of other conjugateswhen TFA was added to mobile phases. BaP sulfates and glucuronidesgenerally coeluted but were partially resolved at 0.1% TFA ina water-methanol gradient. Water-soluble metabolites from culturedhamster embryo fibroblasts (HEF) incubated with [³H]BaP for24 h were chromatographed by h.p.l.c. in a water-methanol gradientwith TFA. BaP glucuronides, consisting of tetraols, triols,quinones, dihydrodiols and phenols eluted as a single peak whichcould be removed by ß-glucuronidase treatment andorganic extraction. BaP sulfates were not detected. The remainingBaP metabolites which were resistant to enzymatic hydrolysis,generally eluted prior to BaP glucuronides suggesting they constitutea family of BaP-GSH derivatives.     

DNA binding and adduct formation of aflatoxin B1 in cultured human and animal tracheobronchial and bladder tissues

DNA binding and adduct formation of aflatoxin B₁ (AFB₁) wasstudied in cultured bladder and tracheobronchial explants fromhuman, monkey, dog, hamster and rat. Explants were exposed to[³H]AFB₁ (1 µM final concentration) in PFMR-4 medium (pH7.4) without serum for 24 h, after which epithetial cell DNAwas isolated by hydroxylapatite chromatography. Binding (µmolAFB₁/mol deoxyribo-nucledetide, mean ± SD) was higherin tracheobronchial tissues (human, 2.2 ± 2.4; rat, 5.7± 2.4; dog, 10.6 ± 6.6; hamster 134.6 ±44.6) than in bladder tissues (human, 1.5 ± 2.3; monkey,2.5 ± 1.1; rat, 3.8 ± 1.1; dog, 5.2 ± 2.3;hamster, 26.2 ± 13.3). These binding levels were notcorrelated with the relative susceptibilities of these speciesto AFB₁ hepatocarcinogenesis, in that the hamster and the dogare insensitive, but exhibited the highest binding, while thesusceptible species, the rat and the monkey, had lower binding.After acid hydrolysis of the isolated DNA, the [³H]AFB₁-DNAadducts were separated by h.p.l.c. In all cases, almost allof the [³H]AFB1-DNA represented addition of AFB₁ to the N⁷ atomof guanine, the major adduct (40–79% of the total) being8, 9-dihydro-8-(N⁵-formyl-2', 5', 6' -triamino-4' -oxo-N⁵-pyrimidyl)-9-hydroxyAFB₁,with minor amounts (7–28%) of 8,9-dihydro-8-(N⁷-guanyl)-9-hydroxyAFB₁.In some cases small amounts (0–8%) of unknown, polar adductscould be detected. It is concluded that, qualitatively, AFB₁-DNAadduct formation by human and animal bladder and tracheobronchilalexplants is similar to that found in other in vitro and in vivoextrahepatic and hepatic systems, but that in vitro bindingdata of AFB₁ to extrahepatic animal tissues can probably notbe used to predict the susceptibility of the human to AFB₁-relatedcardnogenesis in these tissues.     

Catechol-induced alterations in metabolic activation and binding of enantiomeric and racemic 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrenes to DNA in mouse skin

Catechol (1,2-dihydroxybenzene) is a potent co-carcinogen withbenzo[a]pyrene (BaP) and with (?)-7,8-dihydroxy-7, 8-dihydrobenzo[a]pyrene(BaP-7, 8-diol) in mouse skin. The effects of catechol on themetabolic activation of (+)- and (–)- [³H]BaP-7,8-diolsand on epidermal DNA adduct formation of racemic and enantiomeric[³H]BaP-7, 8-diols were examined by applying the tritlated diolsto mouse skin. The major metabolite of the (+)- [³H]BaP-7, 8-diolswas the hydrolysis product of (–)- [³H]-7,8ß-diolsepoxy-9ß,10ß-epoxy-7,8,9, 10-tetrahydroheiizo[a]pyrene (anti-BPDE).This suggests that a peroxyl radical-mediated pathway Is predominantlyresponsible for the epoxidation of this diol. Formation of (–)-anti[³H]BPDEfrom (+)-[³H]BaP-7,8-diol was greater than that of (+)-anti-BPDEfrom (–)-[³H]BaP-7,8-diol Co-application of catechol with[³H]BaP-7,8-diols inhibited epoxidation of the (+) enantiomerto a greater extent than that of the (–) enantiomer. Catecholdecreased the total DNA-binding and the formation of the majoradduct with (+)-[³H]BaP-7, 8-diols metabolites but catecholhad no significant effect on the binding and formation of (+)-anti-[³H]BPDE-deoxyguanosine the major DNA adduct derived from (–)-[³H]BaP-7,8-diolsCo-administration of catechol with (?)-[³H]BaP-7,8-diols increasedthe ratio of (–)- to (+)-[³H]BaP-7, 8-diols major DNAadducts in mouse skin suggesting that catechol selectively inhibitscertain pathways of metabolic activation of (? )-[³H]BaP-7,8-diols Thus, catechol modifies the tumorigenic activity of(?)- BaP-7 ,8.-diol either by alteration of the relative proportionof various hydrocarbon:DNA adducts or by a totally differentas yet unexplored mechanism.