Effect of pretreatment of rats with N-methyl-N-nitrosourea on the repair of O6-methylguanine in liver DNA

The capacity of rat liver to repair O⁶-methylguanine was determinedby measuring the amounts of this base present in DNA after administrationof N-[¹⁴C]methyl-N-nitrosourea (MNU). Chronic pretreatment ofanimals with unlabelled MNU either once weekly for 5 weeks ordaily on weekdays for 14 days had either no marked effect orinhibited (or overloaded) repair activity, respectively. Pretreatmentof rats with single doses of MNU initially decreased O⁶-methylguaninerepair but this effect was lost as the pretreatment intervalincreased and by 40 h, activity had returned to control values.Administration of methylmethanesulphonate for 14 days had noeffect on the removal of O^6-methylguanine produced in liverDNA by [¹⁴C]MNU. None of the pretreatment schedules inhibitedthe removal of 3-methyladenine from liver DNA. These effectsare contrasted with those produced by pretreatment of rats withdimethylnitro-samine (DMN) or 1, 2-dimethylhydrazine which enhancedthe capacity of the liver to remove O⁶-methylguanine producedby single dose of [¹⁴C]DMN.     

The role of formaldehyde in hydrazine-induced methylation of liver DNA guanine

Administration of the hepatotoxin and carcinogen, inorganichydrazine, to rodents results in the formation of 7-methyl-guanineand O⁶-methylguanine in liver DNA; co-administration of [methyl-¹⁴C]methionineor [¹⁴C]formate with the hydrazine labels the methylguanines,suggesting involvement of the 1-carbon pool in the methylationprocess. The present study investigates the proposal that themethylation mechanism involves reaction of hydrazine with endogenousformaldehyde to yield formaldehyde hydrazone, which could bemetabolized to the potent methylating agent diazomethane. Hamsterswere pretreated with methanol, ethanol or cyanamide to alterthe endogenous hepatic aldehyde levels prior to administrationof hydrazine. Formaldehyde levels were refractory to the pretreatments;hepatic acetaldehyde levels were increased, but hydrazine administrationunder such conditions did not result in the formation of ethylatedguanines in DNA. Methanol and ethanol inhibited hydrazine-inducedmethylation of DNA. Hydrazine incubated with liver S9 fractionand calf thymus DNA induced the formation of 7-methylguanineand O⁶-methylguanine when formaldehyde was present in the incubationsystem; substitution of formaldehyde with acetaldehyde in theincubation medium did not result in any detectable alkylationof DNA. Both liver microsomal and cytosolic fractions demonstratedheat-labile activity in supporting the hydrazine-induced methylationprocess. Tetraformyltrisazine, or a similar reaction productof hydrazine and formaldehyde, may be a more important intermediatethan formaldehyde hydrazine in the hydrazine-induced methylationof DNA.     

Carcinogenesis and aging. II. Modifying effect of aging on metabolism of methyl(acetoxymethyl)nitrosamine and its interaction with DNA of various tissues in rats

Young (3 month-old) and old (14 month-old) female outbred ratsreceived a single i.p. dose (13 mg/kg) of N-[¹⁴C]methyl-N-acetoxymethylnitrosamine([¹⁴C]DMN-OAc, which, under these conditions, selectively inducesintestinal tumours. Complete DMN-OAc breakdown occurred within30 min in both young and old rats but exhalation of ¹⁴CO₂ continuedfor over 1 h in young rats and over 3 h in old rats. The highestlevel of methylation in both young and old rats was found inthe DNA of epithelial cells of the colon and in other adjacentabdominal organs (liver and uterus). The initial capacity forexcision of the O⁶-methylguanine from liver DNA was greaterin young animals, but further this DNA adduct was repaired moreefficiently by the liver of old rats. In the DNA of ileal andcolonic enterocytes, O⁶-methylguanine excision was higher inyoung than in old animals. The DNA tertiary structure, measuredby the sedimentation pattern of nucleoids in neutral sucrosegradient, was damaged in old rats, and, to a lesser extent,in young rats. The non-uniform p. ttern of DNA damage in youngand old animals may be associated with differing carcinogeniceffects of DMN-OAc in rats of different ages, a hypothesis whichis currently under test.     

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.     

DNA methylation during chronic administration of 1,2-dimethyl-hydrazine in a carcinogenic regimen

The formation and persistence of O⁶-methylguanine and 7-methylguaninein colon, kidney, and liver DNA were measured over a periodof 25 weeks during 1,2-dimethyl-hydrazine (SDMH) carcinogenesis.Rats were given 14 weekly s.c. injections of 21 mg SDMH/kg bodywt., and methylated guanines in DNA were determined quantitativelyone week after each injection to measure the long-term accumulationof these aberrant bases. No accumulation of either base wasseen in colon DNA, and no O⁶-methylguanine was seen to accumulatein liver DNA. The concentrations of O⁶-methylguanine and 7-methylguaninein kidney DNA did increase with repeated administration of thecarcinogen, but these bases were removed within six weeks afterthe last (14th) injection of SDMH. Large amounts of 7-methylguanineaccumulated in liver DNA over the 14-week treatment period.The concentration of 7-methylguanine in liver and kidney DNAincreased at rates greater than could be accounted for by usingkinetic relationships determined in single-exposure studies.The concentration of O⁶-methylguanine in kidney DNA also increasedat a rate greater than would be expected from calculations basedon the rate of removal following a single administration ofSDMH.     

Effects of carcinogens and partial hepatectomy upon the hepatic O6-methylguanine repair system in mice

The enhanced repair of O⁶-methylguanine from hepatic DNA inrats is a well established phenomenon that can be induced bypretreatment with a variety of hepatotoxic agents as well asby partial hepatectomy. Attempts to induce a similar hepaticresponse by treatment with single doses of aflatoxin B₁ in C57Blmice, single doses of dimethylnitrosamine (DMN) in C57BI orBALB/C mice or by prolonged pretreatment with DMN by additionto the drinking water of C57Bl mice were uniformly unsuccessful.Furthermore, the intense proliferative response induced by two-thirdspartial hepatectomy in C57Bl mice was also apparently incapableof increasing the activity of this repair function. These experiments,in which repair of O⁶-methylguanine has been determined bothin vivo after administration of [¹⁴C]DMN and in vitro usingcrude tissue extracts and a methylated DNA substrate, indicatethe presence of an intergeneric difference between rats andmice with respect to this repair function.     

Dose-response studies of MeIQx in rat liver and liver DNA at low doses

2-Amino-3,8-dimethylimidazo [4,5-f]quinoxaline (MeIQx) is aheterocyclic amine mutagen found in cooked meats and is carcinogenicin mice and rats at high doses (mg/kg body wt). Humans, however,are exposed to low amounts (p.p.b.) in the diet, and the effectscaused by exposure to human equivalent doses of MeIQx have beendifficult to determine accurately. We report on the effect ofMeIQx exposure on liver bioavailability, hepatic DNA bindingand MeIQx persistence in both liver tissue and liver DNA afteracute (24 h), and subchronic (7 day and 42 day) exposures inmale Sprague-Dawley rats. Male Sprague-Dawley rats were administered[2-¹⁴C] MeIQx either by gavage or in the diet for 1, 7 or 42days (1x10^-6mg/kg day up to 3.4x10^-2 mg/kg day dose) and the[2-¹⁴C]MeIQx was measured by accelerator mass spectrometry (AMS).Assessment of the kinetics of hepatic MeIQx DNA adduct formationover 42 days (1.1x10^-4 mg [2-¹⁴C]MeIQx kg daily dose) showsthat steady-state [2-¹⁴C]MeIQx tissue concentrations of 138± 15 pg/g liver and DNA adduct levels of 113 ±10 ag adduct/µg DNA were reached at 14–28 days and28 days respectively. The relationship between administereddose and either hepatic MeIQx DNA adduct levels or MeIQx tissuelevels are linear for the 24 h, 7 day and 42 day exposures.Furthermore, MeIQx adducts persist for at least 14 days afterexposure ceases. These data suggest that bloavailability andDNA adduction by MeIQx increase linearly with increasing dosefor both acute and subchronic exposures. These data also showthat MeIQx DNA adducts are useful in predicting daily exposureand support a linear extrapolation in the risk assessment ofMeIQx. However, the quantitative relationship between DNA adductsand tumor formation will also depend on the specific tissueand the subsequent steps needed for tumor progression.     

Effect of acute doses of 2-acetylaminofluorene on the capacity of rat liver to repair methylated purines in DNA in vivo and in vitro

Male Wistar rats were given various doses of 2-acetylaminofluorene (AAF) at doses of 0.74, 2.22, 6.67, or 20 mg/kg i.p. 24 hr before administration of [14C]dimethylnitrosamine (1 or 2 mg/kg i.p.). Analysis of liver DNA isolated from animals killed 5 hr later showed variations between groups treated with different amounts of AAF in the amounts of 3-methyladenine, 7-methylguanine, and O6-methylguanine (O6-mGua). However, the relative amounts of these products were unchanged by AAF pretreatment except after 20 mg/kg when the reduced O6-mGua:7-methylguanine ratio indicated enhanced O6-mGua repair. Specific enhancement of O6-mGua repair was also found 5 hr after administration of [14C]methylnitrosourea (11.5 mg/kg) to animals pretreated with AAF (20 mg/kg), while the amounts of O6-mGua in liver ribosomal RNA afer [14C]dimethylnitrosamine were unaffected by this AAF dose. Pretreatment of rats with AAF 29 hr earlier increased the capacity of cell-free liver extracts to remove O6-mGua from [3H]methylnitrosourea-methylated DNA in vitro. The increase was detectable after 2.22 mg/kg and reached a maximum 3.5-fold increase after AAF, (60 mg/kg). 7-Methylguanine and 3-methyladenine-DNA glycosylase activities were also increased, but this was independent of the dose of AAF. AAF pretreatment produced a slight (3- to 4-fold) increase in incorporation of [3H]thymidine or labeled one-carbon breakdown products of [14C]dimethylnitrosamine into liver DNA which appeared to parallel in vitro O6-mGua repair enhancement, but the increased [3H]thymidine uptake was statistically significant only after the 60-mg/kg dose.     

Nafenopin-induced rat liver peroxisome proliferation reduces DNA methylation by N-nitrosodimethylamine in vivo

The hypolipidaemic drug nafenopin (NAF) has been shown to enhancethe hepatocarcinogenic effect of N-nitrosodimethylamine (NDMA)and N-nitrosodiethylamine in rats. We have investigated whetherthe NAF-induced peroxisome proliferation in hepatocytes interfereswith NDMA's metabolism and interaction with DNA. Adult maleWistar rats received a single i.p. injection of [¹⁴C]NDMA (2mg/kg) and were killed 4 h later. DNA was isolated from liverand kidney, hydrolysed in 0.1 N HCI and analysed by Sephasorbchromatography. In rats pre-treated with NAF (0.2% in the dietover a period of 3 weeks), the concentration of N7-methylguaninein hepatic DNA (µmol/mol guanine) was 46% below controlvalues. This is probably due to the greater amount of targetDNA, as NAF caused a marked hepatomegaly with a 50% increasein total liver DNA content. Concentrations of N7-methylguaninein kidney DNA were twice as high in NAF-pre-treated animalswhen compared to control rats. This is unlikely to result froma shift in the metabolism of NDMA from liver to other rat tissuessince the time course and extent of the conversion of [¹⁴C]NDMAto ¹⁴CO₂ and ¹⁴C-labelled urinary metabolites were identicalin NAF-treated and control animals. There was no indicationthat NAF inhibits the activity of the hepatic O⁶-alkylguanine-DNAalkyltransferase.     

S-Adenosylmethionine metabolism and DNA methylation in hydrazine-treated rats

The treatment of rats with hepatotoxic doses of hydrazine (NH₂-NH₂)induces the rapid formation of 7-methylguanine and O⁶-methylguaninein liver DNA. The methyl moiety in these reactions might bederived from the cellular S-adenosyl-methionine pool becauseradioactivity administered to these rats as methionine rapidlyappears in the DNA as methylated guanine. An increased incorporationof radioactivity into 5-methylcytosine was previously reportedfollowed by subsequent suppression. This increased radiolabelingof 5-methylcytosine coincided with time of maximal DNA guaninemethylation. To determine the nature of S-adenosyl-methioninemetabolism during the period of DNA methylation induced by hydrazinetreatment, and to determine if the increased radiolabeling of5-methylcytosine at this time reflected an actual increase in5-methylcytosine synthesis, liver DNA synthesis and S-adenosylmethioninelevels and turnover were assayed. Liver S-adenosylmethionineconcentrations varied slightly between control rats and hydrazine-treatedrats during the first five hours after hydrazine ad ministration,and no difference was detectable in the incorporation of administered[³H]methionine into S-adenosylmethionine. Because S-adenosylmethioninespecific radio activity in hydrazine-treated rats was not differentfrom control rats, the previously observed increased radiolabelingof 5-methylcytosine appeared to represent an actual increasein synthesis. This conclusion was supported by finding thatin corporation of radioactive thymidine into DNA was also acceleratedimmediately following hydrazine administration, again followedby a decrease. 5-Methylcytosine synthesis, therefore, appearsto follow DNA synthesis during hydrazine toxicity, and formationof 7-methylguanine and O⁶-methylguanine in liver DNA of hydrazine-treatedrats occurs during a short period of increased DNA synthesisand 5-methylcytosine formation very early in hydrazine toxicity.     

Effect of chronic ethanol diet on the replication, alkylation, and repair of DNA from hepatocytes and nonparenchymal cells following dimethylnitrosamine administration

Chronic alcohol ingestion has been associated with human cancerof the larynx, esophagus, and liver, however the mechanism(s)of ethanol's interaction in either initiation and/or progressionof cancer is unknown. The initiation of chemical carcinogenesisis thought to involve an alteration of the DNA which becomesfixed in the daughter strand following the replication of DNAcontaining promutagenic damage. The purpose of this study wasto investigate the effects of a chronic ethanol diet and acutedimethylnitrosamine administration on the replication, alkylation,and repair of DNA from nonparenchymal cells and hepatocytesof rat liver. DNA replication of nonparenchymal cells, but nothepatocytes, was inhibited in animals on the chronic ethanoldiet. Dimethylnitrosamine exposure reversed this inhibitoryeffect 24 h after exposure. Hepatocyte alkylation and repairof DNA following dimethylnitrosamine administration was unaffectedby the ethanol diet. 7-Methylguanine was lost from both cellpopulations equally during a 72 h time course. In contrast,O⁶-methylguanine was removed much more efficiently in hepatocytes.The nonparenchymal cells had 10 times more O⁶-methylguaninethan hepatocytes 72 h after dimethylnitrosamine administration.Lower concentrations of 7-methylguanine and O⁶-methylguaninewere present in nonparenchymal cells of ethanol treated ratscompared to controls. Although in vitro studies have demonstratedincreased microsomal activation and mutagenesis of dimethylnitrosaminefollowing chronic ethanol ingestion, the present study demonstratesthat in vivo metabolism of dimethylnitrosamine resulting inDNA alkylation is not increased by chronic ethanol exposure.     

Methylation of liver DNA guanine in hydrazine hepatotoxicity: dose-response and kinetic characteristics of O6-methylguanine and formation and persistence in rats

Fischer 344 or Sprague Dawley rats were fasted over night andgiven orally 30–90 mg hydrazine/kg body wt. The presenceof 7-methylguanine and O⁶-methylguanine in liver DNA was demonstrated5 and 24 h after hydrazine administration using two differentanalytical techniques. Methylation levels changed little withdose except for the highest dose (ca. LD₅₀ at which the levelsdoubled. In a time-response study, rats were given 90 mg hydrazine/kgbody wt. and killed 0.25 to 96 h later. Both 7-methylguanineand O⁶-methylguanine were detected quantitatively in liver DNAfrom rats as early as 15 min after hydrazine administration.After maximum levels of methylguanines had formed, 7-methylguaninewas removed from DNA at a rate of {small tilde}50% in 47 h;the half-life of O⁶-methylguanine in liver DNA was {small tilde}13h. Three or four, but not one or two, daily administrationsof 3 mg hydrazine/kg body wt. also produced detectable levelsof 7-methylguanine in rat liver DNA. Neither 7-methylguanineor O⁶-methylguanine was detected in comparable amounts of liverDNA from control animals. The study confirms the observationthat hydrazine administration results in the formation of methylatedguanines in liver DNA.     

Selective repair of methylated purines in regions of chromatin DNA

The distribution of methylated purines in different regionsof liver chromatin DNA has been examined after treating ratswith [¹⁴C]dimethylnitrosamine (2 mg/kg). At different timesafter administration of the carcinogen, liver nuclei were isolatedand fractionated by micrococcal nuclease digestion and low andhigh salt extractions into an active chromatin fraction, twofractions comprising the bulk of the genome, and a nuclear matrixfraction. Regions of active chromatin and nuclear matrix tendedto be methylated more readily than bulk chromatin, with respectto formation of both O⁶-methyl-guanine and N-methyl purines.Removal of both 7-methyl-guanine and 3-methyladenine (by repairand depurination reactions) occurred at a relatively uniformrate in all chromatin fractions. In contrast, repair of O⁶-methylguanineproceeded more rapidly from active chromatin than from bulkchromatin, whereas repair of this lesion from nuclear matrixDNA was much slower than for bulk DNA. Pretreat-ment of ratsfor 4 weeks with non-radioactive dimethylnitros-amine beforethe administration of [¹⁴C]dimethylnitrosamine enhanced therate of repair of radioactive O⁶-methylguanine from all chromatinfractions. Nevertheless the rate of loss of the adduct was stillfaster from active chromatin and slower from matrix DNA thanfrom the bulk of the genome. Since pretreatment also elevatedthe rate of liver DNA synthesis especially in the nuclear matrixfraction, there is an increased probability of the fixationof mutations due to the presence of O⁶-methylguanine in thisselected region of the genome. The implications of this persistentO⁶-alkylation of matrix DNA, and rapid repair of O⁶-alkylguaninein active chromatin for the toxicity and carcinogenicity ofalkylating agents are discussed.     

DNA binding of methyl iodide in male and female F344 rats

The genotoxic potency of methyl iodide was investigated in a DNA binding study. Male and female F344 rats were exposed to 14C-labelled methyl iodide orally or by inhalation in a closed exposure system. DNA adducts were detected in the liver, lung, stomach and forestomach of the exposed animals. [14C]3-Methyladenine, [14C]7-methylguanine and [14C]O6-methylguanine could be identified by a combination of three different methods of hydrolysing DNA and subsequent HPLC or GC/MS analysis. The highest values of methylated guanines were determined in the stomach and forestomach of the animals following both oral and inhalative exposure. These results demonstrate a systemic genotoxic effect of methyl iodide.     

Mode of action of methylating carcinogens: comparative studies of murine and human cells

Murine and human cells (mainly lymphocytes) were methylatedin vitro with either N-[¹⁴C]methyl-N-nitrosourea (MNU) or di-[¹⁴C]methyl sulphate (DMS) and the extents of methylation of DNAat O-6 and N-7 of guanine and N-3 of adenine were determined.The cytotoxic action of MNU was also compared with that of DMS,as assessed by their effects on cell division following stimulationof these lymphocytes in culture by concanavalin A (Con A). Theoverall extent of methylation of the DNA of human cells wasabout 70% of that of murine cells after exposure to MNU andDMS. Mouse cells, responding to Con A in culture, were foundto be much more sensitive to both agents than could be accountedfor by these differences in overall extent of methylation ofthe DNA. Significant differences were found between cells intheir ability to rapidly remove O⁶-methylguanine from DNA. Normalhuman lymphocytes were always proficient, but some human lymphoidlines were deficient in this respect, while all the murine cellstested were deficient, as found in vivo for mouse lymphoid tissues.No correlation has yet been found between the susceptibilityof various mouse strains to the carcinogenic action of MNU andtheir ability to remove methylated bases from DNA. The cytotoxicitystudies, showing that normal human lymphocytes were relativelymore resistant than murine cells to methylation by both MNUand DMS, suggested that the ability to remove O⁶-methylguanine(which is produced in a very low proportion by DMS) was notof prime importance in conferring resistance in this type ofassay.     

Ethanol and dimethylnitrosamine and diethylnitrosamine metabolism and disposition in the rat. Possible relevance to the influence of ethanol on human cancer incidence

Alcohol consumption is associated with an increase in humancancer, notably of the oesophagus. We have found that ethanolwill alter profoundly the distribution of two carcinogenic nitrosaminesin the rat. Small oral doses of dimethylnitrosamine (NDMA) areabsorbed from the portal blood as it passes through the liver,and do not reach the extrahepatic organs. Ethanol, equivalentto a man drinking 1 pint (0.5 1) of beer, prevents this firstpass clearance in the rat and exposes sensitive extrahepaticorgans to this carcinogen. As a consequence the alkylation ofkidney DNA by 35 µg NDMA/ kg body weight was Increased4.6-fold by concurrent administration of 240 mg ethanol/kg,and smaller doses of [¹⁴C]NDMA produced detectable alkylationof kidney DNA only if the rats were given ethanol. Measurementof metabolism of NDMA by liver slices confirmed that this actionof ethanol is the result of inhibition by ethanol of NDMA metabolismin liver (K₁ = 0.5 mM). Comparison of urinary excretion in manand rat suggests that ethanol also inhibits first pass dearanceof NDMA in man. There was no complete first pass clearance ofdiethylnitrosamine (NDEA), but while ethylation of kidney DNAwas decreased by ethanol, that of oesophageal DNA was increasedbetween 1.8- and 4.6-fold. Measurement of the metabolism ofNDEA to CO₂ by liver slices, kidney slices, and oesophagealepithelium suggest that the changes in alkylation of kidneyand oesophageal DNA are the result of selective inhibition ofNDEA metabolism in liver and kidney. The ethylation of oesophagealDNA was greater relative to liver after a small dose than aftera large dose possibly because of the low K_m of the oesophagealmetabolic activating system relative to that in liver and kidney.These results explain experiments showing that concurrent administrationof ethanol increases the carcinogenicity and alters the organsaffected by these nitrosamines. It is tentatively proposed thatthe effect of ethanol on human cancer incidence is mediatedthrough similar influences on the metabolism and dispositionof the nitrosamines to which man is exposed.     

The effects of a single dose of dimethylnitrosamine in the Chinese hamster and the persistence of DNA alkylation products in selected tissues

The acute toxicity and carcinogenicity of dimethyl-nitrosamine(DMN) have been examined in the Chinese hamster (Cricetulusgriseus). A single dose of 20mg/kg or less was not acutely lethalbut at 30 mg/kg only a few animals survived. Liver tumours werefound in all of the animals surviving two doses of 20 mg/kgand so far, 25 months after treatment, in 80% of the animalsgiven a single dose of 20 mg/kg. Altering the diet to sucroselumps and 20% glucose in the drinking water ad libitum for 3days prior to DMN treatment had only a slight effect on acutetoxicity and carcinogenicity. In biochemical studies, DNA wasextracted from several tissues of animals killed 9 h after DMNadministration (20 mg/kg). the amounts of normal and methylatedpurines were determined after mild acid hydrolysis and SephadexG-10 chromatography. The highest level of methylated bases wasfound in the DNA of the liver. Measurement of the persistenceof methylated guanines showed that liver, kidney, and lung wereable to remove the major product 7-methylguanine from DNA whilenone of these tissues had any capacity to remove the pro-mutageniclesion O⁶-methylguanine. These results are discussed in relationto the mechanism of action and tissue specificity of alkylatingcarcinogens in this and other experimental animal systems.     

Formation of O6-methylguanine in regenerating rat liver by N-nitrosomethylbenzylamine is not sufficient for initiation of preneoplastic foci

The DNA methylating activities of N-nitrosodimethylamine (NDMA),an initiator of hepatic -glutamyltranspeptidase-positive foci,and N-nitrosomethylbenzylamine (NMBzA), which does not initiate,were studied in regenerating rat liver. Equimolar doses of ¹⁴C-labelledNDMA and NMBzA (33.5 µmol/kg) were administered to maleSprague-Dawley rats 18 h after partial hepatectomy. NDMA andNMBzA both produced 7-methylguanine and O⁶-methylguanine. Theresults suggest that although the formation of O⁶-methylguaninemay be necessary it is not sufficient for initiation of preneoplasticfoci.     

Methylation status of DNA cytosine during the course of induction of liver cancer in hamsters by hydrazine sulfate

Hydrazine, which is toxic and carcinogenic to rodent liver,has been shown to react with endogenous formaldehyde in theliver to form formaldehyde hydrazone (CH₂=N-NH₂), an alkylatingintermediate that methylates DNA guanine at the N7- and (O⁶-positions.Studies were conducted to investigate the role of chronic hydrazine-inducedhepatotoxicity on DNA maintenance methyiation (formation of5-methyldeoxycytosine) and the development of liver cancer.Male Syrian golden hamsters were given hydrazine sulfate (0,170, 340 and 510 mg/I) in drinking water for 21 months (averagedose 0, 4.2, 6.7 and 9.8 mg/kg body wt hydrazine as the freebase). Hepatotoxicity was evaluated histologically, and regenerativeDNA synthesis and maintenance methyiation were measured as theincorporation of [methyl-¹⁴C]thymidine into DNA and the methylmoiety of [methyl-³H]methionine into 5-methyldeoxycytosine inDNA, respectively. Methylguanines were detected in liver DNAat the first observation time of 6 months of treatment; levelsof these aberrant bases decreased or became undetect-able at14 months, and increased in a dose-related manner for the remainderof the study. DNA adducts persisted in the highest dose groupthroughout the study, repeating the results of a similar studypreviously reported by this laboratory (Bosan et al., Carcinogenesis,8, 439–444, 1987). Linear regression analysis of thymidineand methionine methyl moiety incorporation into liver DNA suggestedimpairment of maintenance methyiation of DNA (5-methyldeoxycytosine)in the middle and high exposure animals. Hepatic adenomas andhepatocellular carcinomas developed in a dose-related mannerand were highly correlated to decreased uptake of radiolabelfrom methionine into DNA 5-methylcytosine. These results arepart of a continuing study on alteration of maintenance methyiationduring hydrazine induction of liver cancer.     

Induction of hprt gene mutations in splenic T-lymphocytes from the rat exposed in vivo to DNA methylating agents is correlated with formation of O6-methylguanine in bone marrow and not in the spleen

The suitability of splenic T-lymphocytes as a substitute tissuefor detection of genotoxic effects induced in vivo by chemicalagents that are organ-specifically activated was tested in ratsexposed to single doses at the potent lung-carcinogen 4-(methylnitrosamino)-1-(3-pyri-dyl)-1-butanone(NNK), acetoxymethylmethylnitrosamine (AMMN) or N-methyl-N-nitrosourea(MNU). NNK, AMMN and MNU methylate DNA most likely via the formationof a methanediazohydroxide ion that decomposes to a methyl diazoniumion.For all three agents, an increase in the levels of O⁶-methylguanineand 7-methylguanine in DNA of rat liver and lung was detectedby reverse phase HPLC and electrochemical detection. Treatmentwith NNK did not result in the formation of O⁶-methylguanineand 7-methylguanine in DNA of bone marrow and spleen, correspondingwith the absence of metabolic activation pathways for this compoundin these tissues. For AMMN formation of both O⁶-methylguanineand 7-methylguanine was detectable in DNA of the spleen, whereasin DNA of bone marrow only very low frequencies of 7-methylguaninewere found at a toxic dose. MNU induced O⁶-methylguanine and7-methylguanine in both spleen and bone marrow. Using splenicT-lymphocytes from the rat no increase above control levelsof the hprt mutant frequencies was found for NNK and AMMN forall exposure levels tested, 32 days after chemical exposure.For MNU a dose-dependent increase in hprt mutant frequency wasfound at exposure levels of 0.097 mmol/kg up to 0.582 mmol/kg.DNA sequence analysis was performed on PCR products of hprtcDNA from 39 MNU-induced 6-thioguanine-resistant T-lymphocyteclones. Single base pair substitutions were found in 25 of thesemutants (64%), GC