Deuterium isotope effect on metabolism of N-nitrosodimethylamine in vivo in rat

The maximal rates of metabolic oxidation of N-nitrosodimethylamine(NDMA) and N-nitrosodimethylamine-d₆ (NDMA-d₆) in viva (V^H andV^D, respectively) have been measured by following ¹⁴CO₂ exhalationin rats after intraperitoneal injection of the two ¹⁴C-labelledcarcinogens at high doses (20 or 40 mg/kg). Complete deuterationof NDMA reduced only slightly the maximal rate of metabolismwhen the two substrates were administered separately (V^H/V^D1.2). However, much larger(4-fold) deuterium isotope effectswere observed when mixtures of NDMA with NDMA-d₆ were injected.These results are tentatively interpreted as evidence that C-Hbond cleavage is not a rate-limiting feature of overall metabolism,but that the complex between NDMA and the principal enzyme(s)metabolizing it in vivo freely equilibrates with unbound substrate.Single, large, intrapentoneal doses of NDMA and NDMA-d₆ produceda similar alkylation of rat liver DNA and also of kidney DNA.However, a small oral dose (54 µg/kg) of NDMA-d₆ produced1/3 less alkylation of liver DNA and 3 times as much alkylalionof kidney DNA as did an equimolar dose of NDMA. The reductionin alkylation of liver DNA correlates well with, and possiblyexplains, the decreased ability of NDMA-d₆ to induce liver tumorsin rats. The associated increase in the alkylation of kidneyDNA suggests that this change is due to a decrease in the amountof nitrosamine removed from the portal blood on the first passthrough the liver.     

Activated neutrophils induce prolonged DNA damage in neighboring cells

We have measured the capacity of highly-purified, paraffin oil-elicitedneutrophils to induce DNA single-strand breaks in a newly establishedplasmacytoma cell line, RIMPC 2304, which was induced by a retroviruscontaining the c-myc and V-Ha-ras oncogenes. This cell lineeffectively repairs DNA damage induced by -irradiation. DNAdamage induced by neutrophils was correlated with the oxidativeburst of the neutrophils. The levels of superoxide anion, H₂O₂and HOCl produced after stimulation of the neutrophils (6 x10⁵/cm³) with the tumor promoter phorbol myristate acetate (100nM) were 33.8 µM, 12.8µM and 1.7 µM respectivelyin 15 mm, and 98 µM, 20 µM and 8.7 µM respectivelyin 90 mm.The results of alkaline elution experiments revealedthat when the same concentration of neutrophils was co-incubatedfor15 min in serum-free medium with an equal number of radioactivelylabeled RIMPC 2304 cells, the latter incurred a level of damagethat approximated that caused by 300 rad equivalents of -irradiationor by a 1-min treatment with 20 µM H₂O₂ at 37C. Damagefrom neutrophils was coincident with the oxidative burst; itwas induced rapidly (within 5 min) but remained high for morethan 90 min. The level of damage achieved was dependent uponthe ratio of neutrophils: target cells and was clearly detectableat ratios as low as 0.25:1. Induction of single-strand breakswas completely inhibited by catalase and partially inhibitedby superoxide dismutase, mannitol, and reduced glutathione butnot by Na azide. Addition of the non-steroidal anti-inflammatorydrug indomethacin either enhanced (at 50 µM) or had noeffect (at 2 µM) on the damage detected. Finally, repairof strand breaks induced by neutrophils was significantly slower(half time 10 min) than that observed for repair of similarlevels of damage induced by H₂O₂ or -irradlatlon (half-times3 min, each). The results indicate that neutrophils cause prolongedDNA damage in neighboring cells. Moreover, they indicate thatalthough H₂O₂ produced in the oxidative burst is an essentialmediator of the damage observed, additional reactive oxygenintermediates including the superoxide anion are also implicated.The data are discussed in relation to the possible role of neutrophilsin chronic inflammation and in pristane-induced plasmacytomaformation in mice.     

Liver cell turnover in rats fed a choline-devoid diet

Liver DNA was labeled in a group of weanling Fischer-344 malerats by placing mini-osmotic pumps, loaded with [methyl-³H]thymidine,under the dorsal skin for 14 days. The animals were then placedon either a cholinesupplemented or a choline-devoid diet, andsubgroups on each diet were killed after 1, 2, 4, 8 and 16 weeks.Liver DNA total and specific radioactivities were determinedin all rats. The results were used to estimate the half-life(t) of liver cells, and the fractional rates of liver cell death(K_d and proliferation (K_p). In rats fed the control choline-supplementeddiet, liver cells were estimated to die at an overall Kd of0.16% per day, and to have a t, h of 439.5 days; K_p was higherin younger than in older rats. In rats fed the choline-devoiddiet, liver cells died at a K_d ranging from 4.82 down to 0.93%per day, as the length of the feeding period increased; correspondingt were 14.3 and 74.6 days. In these rats, K_p was more than sufficientto compensate for cell loss. The results show that liver-celldeath represents a major consequence of feeding a choline-devoiddiet to rats, and that the necrogenic action of the diet isa major factor responsible for the highly increased turnoverof liver cells present in these animals. However, evidence wasobtained that the diet has also a primary mitogenic action,beyond those related to replacement of dead cells, and to cellaccretion due to normal growth of the animals and the liver.     

Metabolism of the carcinogen N-hydroxy-N-2-fluorenylacetamide by rat peritoneal neutrophils

The in vitro metabolism of a locally carcinogenic N-hydroxy-N-2-fluorenylacetamide(N-OH-2-FAA) by rat peritoneal polymorphonuclear leukocytes(PMNL), chiefly neutrophils, elicited with intraperitoneal injectionsof proteose peptone, was examined. At 10⁶ PMNL/ml in media containinghalide (X^–), 0.14 M Cl^– ± 0.1 mM Br^–(without Ca⁺⁺ and Mg⁺⁺), addition of 10 nM phorbol myristateacetate (PMA) resulted in generation of superoxide anion andH₂O₂. Subsequent cetyltrimethylammonium Cl^– (Cetac) additionat 0.002% effected myeloperoxidase (MPO) activity release. PMNLtreated with PMA and/or Cetac did not metabolize N-OH-2-FAA(30 µM). However, 1–2 pulses of H₂O₂ (50 µM)after Cetac addition resulted in oxidation of N-OH-2-FAA toN-acetoxy-2-FAA (<0.5 µM) and 2-nitrosofluorene (2-NOF)(1–2 µM). In the presence of Br^– 2-NOF wasincreased (3–5 µM). The results are consistent withoxidation of N-OH-2-FAA by MPO/H₂O₂ and MPO/H₂O₂/X^– viatwo pathways: one electron oxidation leading to N-acetoxy-2-FAAand 2-NOF, and X^–-dependent oxidation to 2-NOF. N-Acetoxy-2-FAA(10 µM) incubated with PMNL under similar conditions wasconverted non-enzymatically to 4-OH-2-FAA (5 µM) and enzymaticallyto N-OH-2-FAA (3 µM). In the presence of H₂O₂, smalleramounts of these products were formed. Formation of N-OH-2-FAAwas prevented by paraoxon (0.1 mM) suggesting O-deacetylaseactivity. However, accountability for N-acetoxy-2-FAA decreasedwith time, presumably because of binding to cellular macromolecules.With H₂O₂ addition, 2-NOF (10 µM) was converted to 0.5or 0.25 µM 2-nitrofluorene by active PMNL or heat-inactivatedcell lysates, respectively. Low recoveries of 2-NOF were alsoattributed to binding. The results suggest that PMNL may beinvolved in activation of the carcinogenic N-arylhydroxamicacids in vivo.     

Relationship of oxidative damage to the hepatocarcinogenicity of the peroxisome proliferators di(2-ethylhexyl)phthalate and Wy-14,643

Quantitative comparisons of the time course of biochemical andmorphological changes induced by peroxisome proliferators resultingin low and high incidences of hepatic cancer have not been conductedpreviously under bioassay conditions. [4-Chloro-6-(2,3 xylidino)-2-pyrimidyl-thio]aceticacid (Wy-14,643) at 0.1% in the diet produced a much higherincidence of hepatic cancer in male rats than 1.2% di(2-ethylhexyl)phthalate(DEHP) in the diet. Both diets, however, caused similar degreesof peroxisome proliferation. To investigate this differencein carcinogenicity, H₂O₂-detoxification mechanisms and indicesof oxidative damage were evaluated in male F-344 rats fed 1.2%DEHP or 0.1% Wy-14,643 for up to one year. DEHP or Wy-14,643treatment increased hepatic catalase activity 25% from 8 to365 days. DEHP or Wy-14,643 treatment decreased hepatic glutathioneperoxidase activity by 50% from 8 to 365 days. Glutathione concentrationswere not affected by 151 days of DEHP or Wy-14,643 feeding.The similar effects of DEHP and Wy on H₂O₂ detoxification enzymesand glutathione concentrations suggests that these factors arenot responsible for the widely different carcinogenicities ofWy-14,643 and DEHP. Hepatic vitamin E concentrations were 50%lower in rats receiving Wy-14,643 for 151 days as compared torats fed DEHP or control diets. Lipofuscin, which was containedwithin lysosomes, was increased 3-fold after 39 days of DEHPand remained at this level up to 365 days of treatment. In comparison,lipofuscin was increased 4-fold after 18 days of Wy-14,643 andcontinued to accumulate in a linear manner reaching values 30-foldover controls after 365 days of treatment. DEHP treatment for39–365 days increased the activities of the lysosomalenzymes -fucosidase, ß-galactosidase and N-acetylglucosaminidase50–100%. The same enzyme activities were increased 4-foldafter 39–365 days of Wy-14,643. Lysosomal cathepsin Bactivity was unchanged by DEHP but doubled by 151 and 365 daysof Wy-14,643. Acid phosphatase activity was unchanged by DEHPbut increased by 50% after 151 and 365 days of Wy-14, 643. Inaddition, conjugated dienes were increased (45%) only in ratsreceiving Wy-14,643 for 151 and 365 days. These data show forthe first time that the magnitude and time course of lipofuscindeposition, induction of lysosomal enzymes and conjugated dieneaccumulation, is correlated closely with the degree of carcinogenicity.Wy-14,643-induced decreases in hepatic vitamin E concentrationscould contribute to the observed accumulation of conjugateddienes at later time points. The data suggest that lipofuscinaccumulation is an early biomarker that is quantitatively predictiveof the carcinogenicity of the peroxisome proliferators DEHPand Wy-14,643.     

Biological consequences of DNA damage introduced in bacteriophage PM2 DNA by hydrogen peroxide-mediated free radial reactions

In order to study the biological consequences of DNA damageinduced by H₂O₂-mediated free radical reactions, DNA from bacteriophagePM2 was exposed to H₂O₂, Fe³⁺-citrate and ascorbate either aloneor in combination. Induction of DNA lesions was determined aswell as the biological activity of the phage DNA. Exposure toH₂O₂ alone resulted in max. 0.2 single-strand breaks per molecule;in the presence of Fe₃₊-citrate, the yield was 4-fold higher.Under both conditions no double-strand breaks could be detectedand the biological activity was not diminished. This indicatesthat low levels of single-strand breaks as generated by H₂O₂/Fe³⁺-citratedo not inactivate PM2 DNA. Exposure to ascorbate in the presenceFe³⁺-citrate resulted in extensive induction of single-strandbreaks. However, at ascorbate concentration where 3 single-strandbreaks per molecule were induced, again no double-strand breakscould be detected and the biological activity of the DNA wasnot diminished. At 5 mM ascorbate, single-strand breaks wereabove the detection limit. Under these conditions, 0.02 double-strandbreaks were induced and the biological activity was reducedto 50%. The contribution of double-strand breaks to biologicalinactiva-tion was calculated to be 3%. When PM2 DNA was exposedto H₂O₂ in the presence of ascorbate/Fe³⁺-citrate, a typicalbiphasic dose-effect relationship was observed both for theinduction of double-strand breaks and biological inactivation,suggesting that one or more reactive species sensitive to H₂O₂play a critical role. The OH scavenger t-butanol appeared tobe relatively inefficient in protecting PM2 DNA, which may indicatethat other reactive species than OH, are involved.Our data suggestthat other reactive species than OH, such as the ferryl ion,are involved in H₂O₂-mediated DNA damage induction and biologicalinactivation.     

Failure of the peroxisome proliferator WY-14,643 to induce unscheduled DNA synthesis in rat hepatocytes following in vivo treatment

Peroxisome proliferator hepatocarcinogens lack genotoxic activityin numerous in vitro assays using non-target cells which donot respond with peroxisome proliferation. Therefore, the effectof in vivo treatment with WY-14,643 on DNA repair was quantitatedin rat hepatocytes, the target cell for carcinogenesis. PalmitoylCoA oxidase and carnitine acetyltransferase activities in isolatedhepatocytes were elevated by WY-14,643 (50 mg/kg/day by gavagefor up to 5 consecutive days) and by WY-14,643 (0.1%) or di(2-ethylhexyl)phthalate (DEHP) (1.2%) feeding (for up to 28 days), indicatingperoxisome proliferation had occurred. DNA repair as unscheduledDNA synthesis (UDS) was measured autoradiographically as netnuclear grains following thymidine incorporation in primaryhepatocyte cultures. Treatment of rats with WY-14,643 (gavageor feeding) or DEHP (feeding) did not induce UDS. Addition of2-acetylaminofluorene to replicate cultures demonstrated thatWY-14,643 or DEHP treatment did not prevent repair response.Additional cultures were treated with H₂O₂ (0.8 mM H₂O₂ 3 xat 1-h intervals) to evaluate the ability of UDS to detect anyrepair which may be induced by peroxisomal metabolism. H₂O₂did not induce UDS at this concentration, nor did it prevent2-acetylaminofluorene- induced repair. UDS was, however, observedin a separate experiment using a higher concentration of H₂O₂.In summary, a highly carcinogenic peroxisome proliferator didnot induce UDS in the target cell for carcinogenesis in spiteof peroxisome proliferation following in vivo treatment.     

Effect of selective and non-selective muscarinic blockade on baclofen inhibition of gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine in Wistar rats

The effects of baciofen, a -amino-n-butyic acid receptor B agonist,on gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidineand how its effects areinfluenced by selective (M₁) and non-selective(M₁ and M₂) pharmacological blockade of muscarinic receptorswere investigated in inbred Wistar rats. Rats were given s.c.injections of 8 mg/kg body wt baclofen with and without 0.5mg/kg body wt atropine (non-selective M¹ and M² muscarinic receptorantagonist)or 1.0 mg/kg body wt pirenzepine (selective M¹ muscarinic receptorantagonist every other day after a 25 week carcinogen treatment.At week 52 baclofen significantly decreased the incidence ofgastric cancers. Concomitant treatment with atropine significantlyattenuated the inhibition by baclofen of gastric carcinogenesis,but combined use with pirenzepine had no significant effecton the inhibition by baclofen of gastric carcinogenesis. Baclofenalso significantly decreased the labeling index of the antralomucosa. Baclofen plus atropine attenuated the decrease in thelabeling index of the antral mucosa due to baclofen, but baclofenplus pirenzepine had no significant effect on the labeling index.These results suggest that the inhibition of gastric carcinogenesisby baclofen is medicated through muscarinic receptors and M₂receptors, but not M₁ receptors, are involved in this response.     

Enhancement of low-dose N-2-fluorenylacetamide-induced hyperplastic liver nodules by pre-existing cirrhosis

The potentiating effect of pre-existing cirrhosis on the fonnationof hyperplastic liver nodules and/or foci was investigated byfeeding a low dose (0.008%) of 2-N-fluor-enylacetamide (FAA)in a choline-deficient (CD) diet for 32 weeks to cirrhotic andnon-cirrhotic rats. Liver cirrhosis was induced by feeding therats a CD diet for the preceding 36 weeks. The number and areaof -glutamyltranspeptidase-positive hyperplastic liver nodulesand/or foci were significantly larger in cirrhotic rats exposedto low-dose FAA than in non-cirrhotic rats similarly treated.Hyperplastic liver nodules and/or foci were not observed inrats continuously fed a CD diet alone for 68 weeks (controlcirrhotic rats). The results suggest that cirrhotic liver mightalter the metabolic response to FAA, even at low doses, andlead to enhanced induction of hyperplastic liver nodules and/orfoci.     

5-Azacytidine potentiates initiation induced by carcinogens in rat liver

To test the validity of the hypothesis that hypomethylationof DNA plays an important role in the initiation of carcinogenicprocess, 5-azacytidine (5-AzC) (10 mg/kg), an inhibitor of DNAmethylation, was given to rats during the phase of repair synthesisinduced by the three carcinogens, benzo[a]-pyrene (200 mg/kg),N-methyl-N-nltrosourea (60 mg/kg) and 1,2-dimethylhydrazine(1,2-DMH) (100 mg/kg). The initiated hepatocytes in the liverwere assayed as the -glutamyltransferase (-GT) positive fociformed following a 2-week selection regimen consisting of dietary0.02% 2-acetylaminofluorene coupled with a necrogenic dose ofCCl₄. The results obtained indicate that with all three carcinogens,administration of 5-AzC during repair synthesis increased theincidence of initiated hepatocytes, for example 10–20foci/cm² in 5-AzC and carcinogen-treated rats compared with3–5 foci/cm² in rats treated with carcinogen only. Administrationof [³H]-5-azadeoxycytidine during the repair synthesis inducedby 1,2-DMH further showed that 0.019 mol% of cytosine residuesin DNA were substituted by the analogue, indicating that incorporationof 5-AzC occurs during repair synthesis. In the absence of thecarcinogen, 5-AzC given after a two thirds partial hepatectomy,when its incorporation should be maximum, failed to induce any-GT positive foci. The results suggest that hypomethylationof DNA per se may not be sufficient for initiation. Perhapstwo events might be necessary for initiation, the first causedby the carcinogen and a second involving hypomethylation ofDNA.     

Inductions of oxidative DNA damage and mesothelioma by crocidolite, with special reference to the presence of iron inside and outside of asbestos fiber

Inductions of oxidative DNA damage (oh⁸dG) in vitro and peritonealmesothelioma in rats (F344, female) were compared between crocidolite(CR) and de-ironized crocidolite [DCR, washed by HCl and ethylenediaminetetraacetic acid (EDTA)] to verify the hypothesis that reactiveoxygen species contribute to carcinogenesis, focusing on therole of iron present inside or outside of the CR. The yieldof oh⁸dG was 14.6 oh⁸dG/10⁵ in CR and 30.2 in DCR under simpleincubation with DNA. In the incubation systems added severalchemicals and H₂O₂, OCR induced higher levels of oh⁸dG thanCR. Especially, the addition of Fe₂O₃ and H₂O₂ to OCR increasedoh⁸dG in DNA depending on the Fe₂O₃ concentration, however,this tendency was not observed in the same system of CR. Surprisingly,7 out of 10 rats died within 2 days after the injection of 10mg of Fe₂O₃ following the DCR injection (5 mg/rat), showingnecroses of hepatocytes from the surface of each lobe whereCR and Fe₂O₃ particles had been deposited together. There wasno death in other groups of rats. One year after the i.p. injectionof CR (5 mg/rat, single injection), mesotheliomas were foundin all rats administered OCR and Fe (2 mg/rat, once a week,for 35 weeks), in 4 rats of OCR alone (n = 10), in 5 rats ofCR alone (n = 10) and in none of the rats administered Fe₂O₃alone (n = 10). Therefore, present results indicate that theinduction of oxidative DNA damage changed even when the sametype of asbestos was washed by chemical treatment, and Fe₂O₃promoted the development of mesothelioma which was induced byOCR.     

Cells deficient in oxidative DNA damage repair genes Myh and Ogg1 are sensitive to oxidants with increased G2/M arrest and multinucleation

Oxidative stress generated from endogenous and exogenous sourcescauses oxidative DNA damage. The most frequent mutagenic baselesion 7,8-dihydro-8-oxoguanine and the resulting mismatchedadenine are removed by OGG1 and MYH in mammals. Deficienciesin human MYH or mouse MYH and OGG1 result in tumor predispositionbut the underlying molecular mechanism is not fully understood.To facilitate the study of the roles of MYH and OGG1 in theprotection against oxidative stress, we generated mouse embryonicfibroblast cell lines deficient in these genes. Myh and Ogg1double knockout cells were more sensitive than wild type tooxidants (hydrogen peroxide and t-butyl hydroperoxide), butnot to cis-platinum or -irradiations. The low dosage oxidativestress resulted in more reduction of S phase and increase ofG₂/M phase in Myh^–/–Ogg1^–/– cells thanin wild-type cells, but a similar level of cell death in bothcells. The oxidants also induced more multinucleated cells inMyh^–/–Ogg1^–/– cells than in wild-type,accompanied by centrosome amplification and multipolar spindleformation. Thus, under oxidative stress, Myh and Ogg1 are likelyrequired for normal cell-cycle progression and nuclear division,suggesting multiple roles of Myh and Ogg1 in the maintenanceof genome stability and tumor prevention. Abbreviations: H₂O₂, hydrogen peroxide; MEF, mouse embryo fibroblast; TBH, t-butyl hydroperoxide Received September 26, 2007; revised January 17, 2008; accepted January 26, 2008.     

Autoradiographic studies on in vivo covalent binding of N-hydroxy-2-acetylaminofluorene in rat liver containing {gamma}-glutamyltranspeptidase-positive foci. The effect of the sulfation-inhibitor pentachlorophenol

The role of sulfation in the covalent binding of [ring-³H]-N-hydroxy-2-acetylaminofluorene(N-OH-AAF) in rat liver containing -glutamyltranspeptldase-positive(GGT⁺) foci was studied in vivo by autoradiography. GGT⁺ fociwere induced by initiation with diethylnitrosamine, followedby a selection protocol consisting of a 2-week exposure to 2-aminofluorenein the drinking water and a single administration of CCl₄. Bothsurrounding (‘normal’) liver tissue and, to a lesserextent, GGT⁺ foci covalently bound N-OH-AAF, administered 10days after selection. The sulfation inhibitor, pentachlorophenol(PCP), reduced the covalent binding of N-OH-AAF in cells surroundingthe foci. However, PCP had no effect on binding of radiolabelin GGT⁺ foci. Thus, reduced sulfotransferase activity may contributeto the resistance of GGT⁺ preneoplastic lesions to carcinogencytotoxicity. The results suggest also, that cells in GGT⁺ fociare able to bind N-OH-AAF covalently by a sulfotransferase-independentpathway.     

Recombinant rat and hamster N-acetyltransferases-1 and -2: relative rates of N-acetylation of arylamines and N,O-acyltransfer with arylhydroxamic acids

Genes for the 290 amino acid, 33–34 kDa cytosolic acetyltransferases(NAT1^* and NAT2^*) from rat and hamster were cloned and expressedin Escherichia coli. Active clones were selected by a simplevisual test for their ability to decolorize 4-aminoazobenzenein bacterial medium by acetylation. These recombinant acetyltransferaseswere analyzed for: (i) N-acetyltransferase, which was assayedby the rate of acetyl coenzyme A-dependent N-acetylation of2-aminofluorene (2-AF) or 4-aminoazobenzene (AAB); (ii) arylhydroxamicacid acyltransferase, assayed by N, O-acyltransfer with N-hydroxy-N-acetyl-2-aminofluorene.Both NAT2s showed first order increases in N-acetylation rateswith increasing 2-AF or AAB concentrations between 5 and 100µM, with apparent K_m values of 22–32 and 62–138µM respectively. Although under the same conditions theN-acetylation rates for the two NAT1s declined by >50%, below5 µM 2-AF or AAB, the NAT rate data fit Michaelis-MentenKinetics, and the apparent K_m values were 0.2–0.9 µM.For N, O-acetyltransferase, the apparent K_m values of the NAT1swere 6 µM, while the K_m values of the NAT2s were 20- to70-fold higher. SDS-PAGE/Western blot analysis of the recombinantacetyltransferases gave apparent relative molecular weights(MW_r) of 31 kDa for both NAT1s and rat NAT2 and 29 kDa for hamsterNAT2. Comparable MW_r values were observed for native hamsterliver NAT1 and NAT2 and for rat NAT1 under the same conditions.Although we did not detect NAT2-like activity in rat liver cytosolpreviously, the present data show that the rat NAT2^* gene doescode for a functional acetyltransferase, with properties similarto those of hamster liver NAT2. The data also indicate thatat low substrate concentrations, NAT1 would apparently playthe predominant role in vivo in N-acetylation and N, O-acyltransferof aromatic amine derivatives, including their metabolic activationto DNA-reactive agents.     

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.     

Effect of modifying agents on the phenotypic expression of cytochrome P-450, glutathione S-transferase molecular forms, microsomal epoxide hydrolase, glucose-6-phosphate dehydrogenase and {gamma}-glutamyltranspeptidase in rat liver preneoplastic lesions

The expression of A and P forms of glutathione S-transferase(GST-A and P), two cytochrome P-450 isoenzymes (P-450 PB_3a andP-450 MC₂), microsomal epoxide hydrolase (mEHb), glucose-6-phosphatedehydrogenase (G6PD) and -glutamyltranspeptidase (-GT) was comparedin preneo-plastic liver lesions and background parenchyma ofF344 rats post-treated with butylated hydroxyanisole (BHA),ethoxyquin (EQ) or acetaminophen (AAP). These latter three compoundshave been shown to inhibit hepatocarcinogenesis after initialtreatment with N-ethyl-N-hydroxyethylnitrosamine (EHEN) anda significant decrease in the number of enzymealtered foci andnodules positive for GST-P, GST-A, G6PD and -GT and negativefor P-450 PB_3a, P-450 MC₂ was associated with their administration.Whereas in the foci case the decrease was most prominent fornon-discrete (heterogeneous) type lesions, the results of quantitationof nodules revealed a most significant alteration in the discretehomogeneously staining population. This indicates that BHA,EQ and AAP have the potential to inhibit the growth of the phenotypicallystable lesions thought most likely to be the immediate precursorsof hepatocellular carcinomas. The two anti-oxidants were associatedwith periportal increase of all enzymes investigated, whereasAAP induced GST species and mEHb in the perivenular zone. Irrespectiveof slightly elevated enzyme levels in surrounding parenchyma,mEHb antibody binding levels within lesions showed a reciprocalshift from positive to negative in rats treated with BHA, EQand AAP.     

Pyruvate kinase isoenzymes in altered foci and carcinoma of rat liver

Pyruvate kinase (PK) isoenzymes, rate limiting for the laststeps of glycolysis, were studied in normal rat liver, putativepreneoplastic foci, neoplastic nodules and hepatocellular carcinoma.These lesions were produced by an initiation-promotion protocol:treatment with a single dose of N-nitrosomorpholine (NNM) wasfollowed by feeding diets containing phenobarbital (PB) or -hexachlorocyclohexane(-HCH), or basal diet. PK was demonstrated (i) by immunocytochemistryon histological sections with antibodies specifically directedagainst the L and M₂ isoenzymes, (ii) by electrophoretic separationof isoenzymes in homogenates from liver and larger tumors, and(iii) by electrophoretic separation of isoenzymes in parenchymaland stromal cells isolated from liver and tumors. Immunocytochemistryshowed decreases of L-PK (L-PK-) in hepatocytes of most of thefoci, nodules and carcinomas. Most L-PK-foci showed increasesin -glutamyltransferase (-GT) and epoxide hydrolase (EH). PBor -HCH treatment further decreased expression of L-PK in foci,but not in normal liver. Cells and foci with enhanced L-PK (L-PK⁺)were also found after carcinogen treatment. These did not showincreases of -GT or EH or any distinct morphological alterationswith the exception of some which were basophilic (‘tigroid’)in H and E stained sections. No L-PK⁺ tumors were found. Wecould not demonstrate the M₂-type PK in parenchymal cells ofliver or any of the lesions described above. This isoenzymewas restricted to stromal cells in normal rat liver and in allstages of carcinogenesis as shown by immunohistology and byelectrophoresis of preparations from isolated cell populations.However, stromal cells from hepatocellular carcinomas exhibiteda 3-fold increase of M₂-PK compared with stromal cells fromnormal liver. These results do not support an isoenzyme shiftfrom L to M₂-PK in the course of malignant transformation ofhepatocytes as suggested previously.     

Ring-opened 7-methylguanine nucleotides are resistant to nuclease P1 digestion and good substrates to polynucleotide kinase

Dimethyl sulfate was used to prepare 7-methyl-2'-deoxy-guanosine3'-monophosphate (7-methyl-dGMP), whkh was ring-opened in alkalito 2'-deoxy-N⁵-methyl-N⁵-formyl-2,5,6-triamino4-oxopyrimidine3'-monophosphate (ROM-dGMP). ROM-dGMP was not dephosphorylatedby nuclease P₁ in contrast to normal deoxynucleotides. It wasefficiently 5'-phosphorylated by T₄ polynucleotide kinase. Whenmethylated DNA was alkali-treated and digested with mkro-coccalnuclease, spleen phospbodiesterase and nuclease P₁, ROM-dGMPwas formed and this was labeled with [-³²P]-ATP hi the presenceof polynucleotide kinase. Ring-opening and P₁ treatment appearmethods of choice for ³²P-post-labeUng of 7-alkylguanines inDNA.     

Formation and persistence of O6-(2-hydroxyethyl)-2'-deoxyguanosine in DNA of various rat tissues following a single dose of N-nitroso-N-(2-hydroxyethyl)urea. An immuno-slot-blot study

Rabbit antibodies against O⁶-(2-hydroxyethyl)-2'-deoxyguanosine(O⁶-HEdG) were used to develop a highly sensitive immuno-slot-blotassay for this promutagenic base which enabled the quantitationof 3.6 µmol O⁶-HEdG/mol deoxy-guanosine, correspondingto 5 fmol in a 3-µg DNA sample. This assay was used tostudy DNA hydroxyethylation by N-nitroso-N-(2-hydroxyethyl)urea(HENU) in adult male F344 rats. Initial amounts of O⁶-HEdG 2h after a single i.v. dose of 50 mg/kg were highest in kidney(81 µmol O⁶HEdG/mol deoxyguanosine), followed by lungand liver (67 and 55 µmol/mol dG respectively). Formationof O⁶-HEdG in cerebral DNA was considerably lower (18 µmolO⁶-HEdG/mol deoxyguanosine), probably reflecting delayed crossingof the blood—brain barrier by HENU due to its hydrophilicity.The formation of O⁶-HEdG in liver and kidney was strictly proportionalto dose over a range of 5–50 mg HENU/kg. Repair of O⁶-HEdGwas very rapid in liver (apparent half-life, 12 h), and somewhatslower in kidney and lung (approximate half-life, 40 h and 48h respectively). In contrast, 62% of the initial amount of O⁶-HEdGin cerebral DNA was still present after 7 days. Saturation ofthe hepatic O⁶-alkyl-guanine-DNA alkyltransferase by pretreatmentwith N-nitrosodimethylamine (20 mg/kg) almost completely inhibitedthe removal of O⁶-HEdG, indicating that O⁶-HEdG is predominantlyrepaired by this repair enzyme.     

Studies on the antimutagenic activity of ascorbic acid in vitro and in vivo

The possibility that ascorbic acid, as a nucleophile, may inhibit mutagenicity induced by electrophilic metabolites of N-nitrosocompounds was examined. In vitro data are presented to showthat ascorbic acid does not decrease the mutagenicity of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in a modified Ames bacterialmutagenicity system if deionized water is used to prepare theincubation medium. However, ascorbic acid prevents the mutagenicityof MNNG in vitro if added to bacteria in a medium prepared witheither sterile tap water or deionized water and Cu²⁺ ions andthat this an-timutagenic response is blocked by EDTA. Additionalin vitro experiments suggest that when ascorbic acid and Cu²⁺ions are mixed in aqueous solution, H₂O₂ and free radicals derivedfrom H₂O₂ are formed and these compounds may deactivate N-nitrosocompounds. In vivo data are presented to show that ascorbicacid supplementation to guinea pigs (2000 mg/kg body weight/day)has no effect on the mutagenicity of N-nitrosodimethylamine,MNNG, N-methyl-nitrosourea and streptozotocin using the intrahepatichost-mediated bacterial mutagenicity assay. Additional in vivostudies demonstrate that simultaneous oral administration ofascorbic acid prevents the mutagenicity that follows the in-tragastricnitrosation of aminopyrine by nitrite while dietary pre-treatmentwith ascorbic acid does not. These findings suggest that ascorbicacid can block the intragastric formation of mutagenic N-nitrosocompounds but that ascorbic acid has no effect on mutagenicityof N-nitroso compounds once they are formed.