In vivo and in vitro interaction of 1,2-dichlorobenzene with nucleic acids and proteins of mice and rats.

Twenty-two hours after i.p. injection into male Wistar rats and BALB/c mice, 1,2-dichlorobenzene (1,2-DCB) was covalently bound to DNA, RNA, and proteins of liver, kidney, lung and stomach. The covalent binding index to liver DNA was typical of carcinogens classified as weak initiators. The enzyme-mediated in vitro interaction of 1,2-DCB with calf thymus DNA of synthetic polyribonucleotides was carried out by a microsomal mixed-function oxidase system and microsomal GSH-transferases, which seemed to be effective only in liver and lung of rat and mouse. Cytosolic GSH-transferases played a minor role in 1,2-DCB bioactivation. The latter finding provides the first evidence of 1,2-DCB genotoxicity in mammalian cells. The type of halide, the number of halosubstituents and their spatial disposition on the benzene ring are the major determinants of halobenzenes activability to intermediate(s) capable of interacting covalently with DNA and other macromolecules in biologic systems.     

Chloroform bioactivation leading to nucleic acids binding.

Chloroform was bound covalently to DNA, RNA and proteins of rat and mouse organs in vivo after i.p. injection. Covalent Binding Index values of rat and mouse liver DNA classify chloroform as a weak initiator. Labelings of RNA and proteins from various organs of both species were higher than that of DNA. In an in vitro cell-free system, chloroform was bioactivated by cytochrome P450-dependent microsomal fractions, by cytosolic GSH-transferases from rat and mouse liver, and particularly by the latter enzymes from mouse lung. This observation suggests that GSH plays a role in the binding of chloroform metabolites to DNA. The presence of both microsomal and cytosolic enzymatic systems in the standard incubation mixture generally led to an additive or synergistic bioactivating effect for rat and mouse, respectively.     

Covalent binding of the carcinogen trichloroethylene to hepatic microsomal proteins and to exogenous DNA in vitro

Studies were carried out on the in vitro covalent binding of the carcinogen trichloroethylene (TCE) to liver microsomal preparations and to exogenous DNA. The binding of TCE to liver microsomal proteins of male C57BL/6 X C3H/He F1 (hereafter called B6C3F1) hybrid mice, a species and strain susceptible to TCE-induced liver tumorigenesis, was 46% higher than that of [14C]TCE to microsomal proteins from male Osborne-Mendel rats, a species and strain resistant to TCE-induced hepatocellular carcinoma. The in vitro binding of [14C]TCE to liver microsomal proteins was 37% higher for male B6C3F1 mice; female B6C3F1 mice that have been reported to show a lower incidence of TCE-induced hepatocellular carcinoma than do males. Microsomal proteins from the lung, stomach, and kidney of B6C3F1 hybrid mice also metabolized TCE, as indicated by the covalent binding of [14C]TCE to microsomal proteins from these organs. For rats the binding of TCE to liver microsomal proteins of Sprague-Dawley animals was higher than that of Osborne-Mendel and Fischer 344 rats. Incubation of [14C]TCE with salmon sperm DNA in the presence of microsomal preparations from B6C3F1 hybrid mice resulted in covalent binding of [14C]TCE to DNA. This binding was much higher in the presence of microsomal proteins from male rather than female mice. The binding to DNA and protein was enhanced by in vivo phenobarbital administration. The effects of 1,2-epoxy-3,3,3-trichloropropane on the covalent binding of [14C]TCE to protein and DNA were also examined.     

1,1,2-Trichloroethane: evidence of genotoxicity from short-term tests

At 22 hr after ip injection into adult male Wistar rats, 1,1,2-trichloroethane was covalently bound to DNA, RNA and proteins of the liver, kidney, lung and stomach, as has been found with various weakly carcinogenic halo compounds. The extent of interaction of 1,1,2-trichloroethane with mouse liver DNA was much higher than that with rat liver DNA. This result provides evidence of a correlation between adducts formation and species susceptibility to hepatocarcinogenesis (only the mouse is sensitive to the oncogenic effect of this compound). Interaction with DNA mediated by murine liver microsomes occurred in vitro. No particular differences between the two species were found. In vitro binding was enhanced (approximately 5-fold) by pretreatment in vivo with phenobarbitone but was suppressed by addition of 2-diethylamino-ethyl-2,2-diphenylvalerate X HCl in vitro. Cytochrome P-450 was, therefore, involved in the interaction process. Glutathione suppressed the microsome-mediated interaction, acting as a "scavenger" of reactive intermediate(s). Murine lung microsomes were less effective bioactivators than liver microsomes for the interaction with DNA and microsomal RNA, but not microsomal protein. Kidney and stomach microsomes were ineffective, as were cytosolic fractions from all of the assayed organs of the two species. The extent of in vitro interaction of 1,1,2-trichloroethane with synthetic polyribonucleotides was of the same order of magnitude as that with DNA. The results represent further clear evidence for genotoxicity of 1,1,2-trichloroethane.     

Inactivation of DNA-binding metabolites of benzo[a]pyrene and benzo[a]pyrene-7,8-dihydrodiol by glutathione and glutathione S-transferases

The binding to DNA of reactive metabolites of trans-7,8-dihydro-7,8-dihydroxybenzo[a]pyrene(BP-7, 8-diol) was studied following the incubation of tritiatedbenzo[a]pyrene (BP) and BP-7, 8-diol with nuclei from liversof 3-methyl-cholanthrene-treated rats. Binding was inhibitedto a small extent by glutathione (GSH) alone and to a much greaterextent by GSH and cytosol or purified GSH-transferases B andE. In this respect GSH-transferases A and C were also active,but less so. Inhibition of binding of BP-7,8-diol metabolitesto DNA mediated by GSH-transferases was associated with theformation of GSH conjugates. The extent of inhibition of bindingwas similar in incubations of nuclei alone, nuclei and rat livermicrosomes, and calf thymus DNA and rat liver microsomes. Thisindicates that reactive metabolites of BP-7, 8-diol, formedeither by nuclei or microsomes, are readily accessible to solubleGSH-transferases. GSH and cytosol were also active in inhibitingDNA-binding of reactive metabolites from 9-hydroxybenzo[a]pyrene(9-OH-BP). Thus, in the rat hepatocyte GSH and GSH-transferasesmay be important in protecting DNA from electrophilic attackby reactive BP-7, 8-diol and 9-OH-BP species.     

In vivo and in vitro binding of epichlorohydrin to nucleic acids

Epichlorohydrin (EC) binds to macromolecules of biological relevance in vivo: DNA is less labelled than RNA and proteins, rat organs interact more than mouse organs, stomach is the most labelled organ with liver, kidney and lung involved in decreasing order. Based on the Covalent Binding Index (CBI), EC is a weak-moderate oncogen, just as other chlorinated hydrocarbons such as 1,2-dichloroethane and carbon tetrachloride. An interaction of EC with nucleic acids (DNA and polyribonucleotides) occurs also in vitro. It is mediated either by chemical reactivity per se of the molecule (near-UV (NUV) irradiation does not photoactivate EC) and by enzymatic (microsomal and/or cytosolic) fractions, whose relative effectiveness is variable in relation to the organ tested. The best substrates for interaction are poly(G) and poly(A) when using microsomal and cytosolic fractions, respectively, whereas the labelling of double-stranded DNA is always lower. On the whole, the picture of enzyme (microsome + cytosol)-mediated in vitro interaction is similar to the pattern of in vivo binding, with the exception of rat stomach enzymes which are inactive in vitro.     

Species and organ differences in DNA adduct formation and repair after treatment with 4-hydroxyaminoquinoline 1-oxide

4-Hydroxyaminoquinoline 1-oxide (4HAQO) demonstrates obvious organotropic and species specificity in its carcinogenesis and the present investigation concerns 4HAQO DNA adduct formation and repair as studied in various organs of four animal species (rats, mice, guinea pigs and hamsters). Three hours after an iv injection of 10 mg per kg body weight of tritium-labeled 4HAQO, the major organs were removed and used for assessment of label incorporation in the DNA. The results showed that the DNA binding levels generally correlated well with the reported species and organ specificity of 4HAQO tumorigenesis. For example, rats showed highest DNA binding in the pancreas and kidney, major target organs. The levels of DNA binding in the liver were invariably low in all 4 animal species, in agreement with the lack of hepatocarcinogenicity associated with 4HAQO exposure. A clear relationship between DNA adduct formation and carcinogen dose was also found after treatment of mice with 4HAQO at doses of 1, 5, 10 and 20 mg per kg body weight in all tissues (pancreas, kidney and lung) except for the liver. Comparison of DNA repair processes in rats, a highly susceptible species, and hamsters, a resistant species in terms of 4HAQO carcinogenicity, revealed highest formation and slowest removal of adducts in the target organs of the rat. In the hamster organs and the rat lung and liver, DNA adduct formation was generally low and in the case of elevation in the initial phase, quickly removed.     

Species and Organ Differences in DNA Adduct Formation and Repair after Treatment with 4-Hydroxyaminoquinoline 1-Oxide

4-Hydroxyaminoquinoline 1-oxide (4HAQO) demonstrates obvious organotropic and species specificity in its carcinogenesis and the present investigation concerns 4HAQO DNA adduct formation and repair as studied in various organs of four animal species (rats, mice, guinea pigs and hamsters). Three hours after an iv injection of 10 mg per kg body weight of tritium-labeled 4HAQO, the major organs were removed and used for assessment of label incorporation in the DNA. The results showed that the DNA binding levels generally correlated well with the reported species and organ specificity of 4HAQO tumorigenesis. For example, rats showed highest DNA binding in the pancreas and kidney, major target organs. The levels of DNA binding in the liver were invariably low in all 4 animal species, in agreement with the lack of hepatocarcinogenicity associated with 4HAQO exposure. A clear relationship between DNA adduct formation and carcinogen dose was also found after treatment of mice with 4HAQO at doses of 1, 5, 10 and 20 mg per kg body weight in all tissues (pancreas, kidney and lung) except for the liver. Comparison of DNA repair processes in rats, a highly susceptible species, and hamsters, a resistant species in terms of 4HAQO carcinogenicity, revealed highest formation and slowest removal of adducts in the target organs of the rat. In the hamster organs and the rat lung and liver, DNA adduct formation was generally low and in the case of elevation in the initial phase, quickly removed.     

In vivo and in vitro binding of benzene to nucleic acids and proteins of various rat and mouse organs

Benzene binds to macromolecules of various organs in the rat and mouse in vivo. Labelling of RNA and proteins is higher (1 order of magnitude) than DNA labelling, which is low in many organs (liver, spleen, bone marrow and kidney), and negligible in lung; no difference between labelling of rat and mouse organs was found. The covalent binding index (CBI) value was about 10, i.e. typical of genotoxic carcinogens classified as weak initiators. In vitro binding of benzene to nucleic acids and proteins is mediated by hepatic microsomes, but not by microsomes from kidney, spleen and lung, or by cytosol from whatever organ. Nucleic acid binding can be induced by pretreatment with phenobarbitone (PB) and suppressed in the presence of SKF 525-A, of cytosol and/or GSH or of heat-inactivated microsomes. Labelling of exogenous DNA is low and is similar in the presence of rat or mouse microsomes in agreement with the low interaction with DNA measured in vivo.     

Tissue distribution of DNA adducts in CDF1 mice fed 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ)

Male and female CDF1 mice were administered a single oral dose of 3 mumol of the food mutagens 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) or 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and killed 24 h later. DNA was isolated from the livers, lungs, kidneys, colon and forestomach and analysed by 32P-postlabelling for the presence of IQ and MeIQ adducts. Several adduct-enrichment procedures were investigated, including ATP-deficient labelling conditions, butanol extraction and nuclease P1 digestion, and only the ATP-deficient procedure was found to produce the same adduct pattern on polyethyleneimine--cellulose TLC as the standard procedure. Up to nine adduct spots were detected in liver DNA from IQ-treated mice, two of which were not detected in other tissues. The levels of binding in both male and female mice were in the order liver greater than kidney greater than colon greater than forestomach greater than lung. Analysis of DNA from MeIQ-treated mice revealed the presence of up to seven adducts, one of which was detected in liver but not in other tissues. The relative order of DNA binding was kidney greater than liver greater than or equal to colon greater than forestomach greater than lung. As dietary feeding of IQ induces liver, lung and forestomach tumours, and MeIQ induces liver and forestomach tumours in this mouse strain, these binding levels do not correlate with the susceptibility of the organs to carcinogenesis induced by these compounds; the results may indicate the importance of additional factors in determining organ specificity of carcinogenicity.     

Susceptibility to aflatoxin B1-induced carcinogenesis correlates with tissue-specific differences in DNA repair activity in mouse and in rat

To investigate the mechanisms responsible for species- and tissue-specific differences in susceptibility to aflatoxin B(1) (AFB(1))-induced carcinogenesis, DNA repair activities of nuclear extracts from whole mouse lung and liver and rat liver were compared, and the ability of in vivo treatment of mice with AFB(1) to alter repair of AFB(1)-DNA damage was determined. Plasmid DNA containing AFB(1)-N(7)-guanine or AFB(1)-formamidopyrimidine adducts were used as substrates for the in vitro determination of DNA repair synthesis activity, detected as incorporation of radiolabeled nucleotides. Liver extracts from CD-1 mice repaired AFB(1)-N(7)-guanine and AFB(1)-formamidopyrimidine adducts 5- and 30-fold more effectively than did mouse lung, and approximately 6- and 4-fold more effectively than did liver extracts from Sprague-Dawley rats. The susceptibility of mouse lung and rat liver to AFB(1)-induced carcinogenesis correlated with lower DNA repair activity of these tissues relative to mouse liver. Lung extracts prepared from mice treated with a single tumorigenic dose of 50 mg/kg AFB(1) i.p. and euthanized 2 hours post-dosing showed minimal incision and repair synthesis activities relative to extracts from vehicle-treated mice. Conversely, repair activity towards AFB(1)-N(7)-guanine damage was approximately 3.5-fold higher in liver of AFB(1)-treated mice relative to control. This is the first study to show that in vivo treatment with AFB(1) can lead to a tissue-specific induction in DNA repair. The results suggest that lower DNA repair activity, sensitivity of mouse lung to inhibition by AFB(1), and selective induction of repair in liver contribute to the susceptibility of mice to AFB(1)-induced lung tumorigenesis relative to hepatocarcinogenesis.     

Vinylidene chloride: changes in drug-metabolizing enzymes, mutagenicity and relation to its targets for carcinogenesis

Results of various studies have shown that male Swiss Webstermice are more susceptible to toxic effects of vinylidene chloride(VDC.) than are females of the same mouse strain, females andmales of the C57BL mouse strain, Chinese hamsters and rats.The main targets of toxicity are kidney and liver. The kidneyof male Swiss Webster mice is the only organ where VDC unambiguouslyinduces tumours. In the present study we have investigated theability of NADPH-foritifed postmitochondrial supernatant fractions(S-9 mix) of kidney and liver from susceptible and non-susceptibleanimals to activate VDC to a bacterial mutagen. The followingsequence of activating potencies was observed: mouse liver (bothstrains and sexes) and Chinese hamster liver > rat liver> human liver > Chinese hamster kidney > kidney frommale mice of both strains > kidney from rats and female mice.The last two preparations only occasionally showed weak activationof VDC. Addition of purified micro somal epoxide hydrolase toS-9 mix did not affect the mutagenicity of VDC; addition ofglutathione reduced the mutagenicity up to 50%. Pretreatmentof animals (male rats, male and female Swiss Webster mice) withVDC did not potentiate the ability of the subcellular preparationsto activate this compound. In fact, in some cases, a weakeractivation was observed. Following this treatment, microsomal7-ethoxy-coumarin O-dealkylase was decreased in mouse kidneyand in rat liver. The enzyme was not affected in mouse liverand was not measurable in rat kidney. Microsomal epoxide hydrolaseactivity (with styrene 7,8-oxide as substrate) was not affectedin mouse liver and rat kidney. In the kidney of male mice treatedwith a high concentration of VDC, epoxide hydrolase activitywas decreased initially, but after longer treatment, in somecases a weak increase above control was noticed. A strongerincrease in activity of epoxide hydrolase was observed in therat liver and the kidney of female mice. Cytosolic glutathionetransferase activity (with 2,4-dinitrochioro-benzene as substrate)was not affected by the VDC treatment in the liver of male mice,but was decreased in the kidney of male mice, and was elevatedin the kidney and liver of rats and of female mice. The differenteffects of VDC on this enzyme may be one of the reasons forthe differences in susceptibility towards the toxic and carcinogenicactions of this compound in different species, strains and sexes.     

Species comparison of acrylonitrile epoxidation by microsomes from mice, rats and humans: relationship to epoxide concentrations in mouse and rat blood

Acrylonitrile (ACN) has been shown to cause tumors of the brain, stomach and Zymbal's gland in rats in several bioassays, but it has not been tested in other species. The carcinogenic risk of humans exposed to ACN is unclear. ACN is metabolized in the liver to 2-cyanoethylene oxide (CEO), which is believed to be the proximate or ultimate carcinogenic species. Therefore, the kinetics of CEO formation were studied with liver and lung microsomes from mice and humans using a GC-MS assay for CEO, and the data were compared with previously obtained kinetic parameters for rat microsomal enzymes. The rate of CEO formation by human liver microsomes was comparable to that of rat liver microsomes, but less than that of mouse liver microsomes. Liver microsomes produced more CEO than lung microsomes with all three species. CEO formation by microsomes from mice was approximately 4 times greater than that by microsomes from rats or humans, suggesting that mice would have higher CEO concentrations in blood than rats after ACN exposure. However, after oral administration of ACN, the concentration of CEO in mouse blood was one-third that in rat blood at all doses and time points examined. These results show that CEO circulates via the blood, providing exposure to distant sites. The blood concentrations of CEO do not appear to correlate with rates of microsomal CEO formation. This suggests that species differences in the detoxication of CEO may play an important role in determining circulating CEO concentrations and distant organ exposure.     

In vivo binding of 3,3'-dichlorobenzidine to rat and mouse tissue DNA

The kinetics of total DNA adducts were compared in the liver, bladder epithelium and small intestinal epithelium of rats and mice following a single oral dose (100 mg/kg) of 3,3'-dichlorobenzidine [( 14C]DCB). Peak DNA binding (expressed as pmol DCB bound/mg DNA) in rat tissues was 153.5, 144.8 and 36.9 in the intestine, bladder and liver, respectively, whereas in mouse tissues, the binding was 72.5, 58.2 and 55.8, respectively. In either species, the half-life of the DNA adducts in the liver (13.5 and 13.8 days in rats and mice, respectively) was comparable to that in the bladder epithelium (14.8 and 12.7 days in rats and mice, respectively) but longer than that in the intestinal epithelium (5.9 and 4.7 days in rats and mice, respectively). Peak total DCB binding in hepatic but not intestinal or bladder epithelial DNA correlated positively with total urinary DCB metabolites. In vitro, mouse hepatic S9 was 57% more active in catalyzing the formation of DNA-binding derivatives of DCB, in parallel with the higher in in vivo maximum hepatic DNA binding in mice than in rats. Thus, a single oral dose of DCB in rats and mice leads to extensive binding of the chemical to tissue DNA, with the rate of removal of the adducts not differing between target and non-target tissues.     

Formation and persistence of a DNA adduct in rodents treated with N-nitrosopyrrolidine

The rate of formation and the persistence of an exocyclic guanine adduct formed in DNA of rodents treated with various doses of N-nitrosopyrrolidine (NPYR) have been determined. NPYR is hepatocarcinogenic to the rat and forms a covalent adduct in liver DNA; this adduct was recently identified as 2-amino-6,7,8,9-tetrahydro-9-hydroxypyrido[2, 1-f]purine-4[3H]-one. Dose-dependent amounts of adduct formed in liver, kidney and lung DNA of rats, hamsters and mice given oral doses (56-900 mg/kg body wt) of NPYR. The persistence of the adduct in DNA after administration of low doses of NPYR to rats was greatest in the target organ, i.e. the liver; at high doses of NPYR, adduct levels in DNA changed little over a period of at least 72 h. In the hamster, in which NPYR is carcinogenic to the lung but apparently not the liver, the adduct level in liver DNA was an order of magnitude greater than in lung or kidney DNA for a dose of NPYR of 225 or 900 mg/kg body wt; persistence of the adduct in lung DNA was only slightly longer than in liver DNA. The formation and persistence of the 7,8-pyridoguanine adduct in the rat appeared to be consistent with the organotropy of this carcinogen, but this was not true for the hamster, a species that seems to be more resistant to induction of liver and kidney cancer by this carcinogen. Imidazole, an inhibitor of microsomal amine oxidase, and disulfiram, an inhibitor of aldehyde dehydrogenase, decreased metabolic activation of NPYR to an alkylating intermediate; inducers and inhibitors of cytochrome P450 monooxygenases had little effect on the metabolic activation of NPYR to an alkylating agent.     

Tissue and species specificity of the microsomal metabolism of N-nitrosomethylbenzylamine

The microsomal activation of N-nitrosomethylbenzylamine (NMBzA) by oxidation at the methylene carbon atom was examined in various organs of a number of species to determine the role of metabolism in the organ-specificity of tumour induction by NMBzA. In Sprague-Dawley rats, NMBzA was metabolized by microsomes from liver, lung and oesophageal mucosa. In Fischer F-344 rats and in rabbits, metabolic activity was present in both liver and oesophageal mucosa, the only tissues studied in those species. In contrast, in Syrian hamsters and in BALB/cBYJ mice, no NMBzA metabolism was detectable in the oesophagus, but it occurred at relatively high rates in liver, lung and kidney. The forestomach mucosa exhibited undetectable levels of activity in all species except the hamster in which it was present at a very low level. In human oesophageal mucosal microsomes from six patients, rates of metabolism of NMBzA were either undetectable or approximately 70 times lower than those in the Sprague-Dawley rats. A comparison of NMBzA metabolism in the different species with the known carcinogenicity of the nitrosamine in rats and rabbits, and our preliminary data on the acute toxicity of NMBzA in hamsters and mice suggests that, in the oesophagus at least, metabolic activation of NMBzA is necessary to elicit its toxic and/or carcinogenic effect. However, in the liver, which in all species has high metabolic activity but which is also resistant to the toxic and carcinogenic effects of NMBzA, other factors besides metabolic activation must be involved.     

Quantitation of microsomal alpha-hydroxylation of the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is activated to DNA alkylating species via two different alpha-hydroxylation pathways. Methylene hydroxylation leads to DNA methylation, whereas methyl hydroxylation yields DNA pyridyloxobutylation. We have developed a high-pressure liquid chromatography assay utilizing radiochemical detection that permits the determination of the extent of metabolism through each pathway in microsomal preparations. Levels of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) were used to measure the extent of methyl hydroxylation, whereas levels of the aldehyde, 4-oxo-1-(3-pyridyl)-1-butanone (OPB), were used to quantify the extent of methylene hydroxylation. Incubations of [5-3H]NNK with microsomes and cofactors were conducted in the presence of 5 mM sodium bisulfite to trap the reactive OPB. The inclusion of bisulfite did not affect the rate of NNK metabolism. Trapping the aldehyde also inhibited its further oxidation to the corresponding acid or reduction to HPB. Furthermore, the conversion of HPB to OPB made only a minor contribution to the OPB levels under our incubation conditions. Analysis of incubation mixtures containing [5-3H]NNK, cofactors, and either A/J mouse liver or lung microsomes demonstrated that OPB was a significant metabolite of NNK. The OPB:HPB ratio was greater in liver (1.5) than in lung (0.2-1) microsomal preparations. Apparent Km values for OPB and HPB formation in lung microsomes were 23.7 and 3.6 microM, respectively, whereas the corresponding values for liver microsomes were 19.1 and 73.8 microM, respectively. These data are consistent with the involvement of more than one cytochrome P-450 isozyme in the activation of NNK to DNA reactive species.     

Binding of metabolites of cyclophosphamide to DNA in a rat liver microsomal system and in vivo in mice

The stability of phosphoramide mustard, a metabolite of cyclophosphamide was studied at pH 7.2 and 37 degrees C using 31P nuclear magnetic resonance. The phosphorus signal of phosphoramide mustard disappeared with a half-life of 8 min indicating rapid conversion to other species. The final product, inorganic phosphate, appeared with a half-life of 105 min indicating that phosphoramide mustard was easily dephosphoramidated. A rat liver microsomal system was used to study the binding of [chloroethyl-3H]cyclophosphamide to DNA. DNA was hydrolyzed in 0.1 N HCl:0.5 N NaCl at 80 degrees C for 20 min, conditions known to convert phosphoramide mustard to nornitrogen mustard with liberation of the phosphoramide residue. After such treatment three adducts were detected by high-performance liquid chromatography using several elution systems. They were all 7-substituted guanine adducts of nornitrogen mustard; two were monoalkylation products with an intact [N-(2-chloroethyl)-N-[2-(7-guaninyl)ethyl]amine] or an hydroxylated mustard arm [N-(2-hydroxyethyl)-N-[2-(7-guaninyl)ethyl]amine]; the third adduct was a cross-linked product [N,N-bis [2-(7-guaninyl)ethyl]-amine]. The relative abundance of these adducts depended on the length of the microsomal incubation. After 2 h, N-(2-chloroethyl)-N-[2-(guaninyl)ethyl]amine was the main product but after 6 h N-(2-hydroxyethyl)-N-[2-(7-guaninyl)ethyl]amine was most abundant, and at this time the cross-linked product represented 12% of the total adducts. The adducts in DNA depurinated readily and after 24 h at pH 7.0 and 37 degrees C 70% of them had been liberated. The rate of depurination was decreased in the presence of 0.5 N NaCl. After short-term depurination in 0.1 N HCl at 25 degrees C the primary alkylating species was phosphoramide mustard rather than nornitrogen mustard. In in vivo studies mice were given injections i.p. of 100 microCi of cyclophosphamide. Maximal levels of radioactivity had been incorporated into DNA between 2-7 h after injection; the specific activity of DNA from the kidney and lung exceeded that from the liver. While the level of radioactivity found in kidney DNA was rapidly reduced the rate of fall was lower in the lung. Between 24 and 72 h the specific activity of lung DNA exceeded that of kidney and liver DNA by a factor of 3:8. Lung is the principal target tissue for tumor formation in mice after an i.p. injection.     

DNA methylation by N-nitrosomethylbenzylamine in target and non-target tissues of laboratory rodents. Comparison with carcinogenicity

Following oral or systemic (subcutaneous) administration to rats,N-nitroso -methylbenzylamine (NMBzA) causes a high incidence of oesophageal tumours. Methylation of DNA purines by a single oral dose of [14C-methyl]-NMBzA was most extensive in the oesophagus, followed by liver, forestomach and lung. After a single intravenous injection, alkylation levels were also highest in oesophageal DNA, followed by liver, lung and forestomach. These differences in the extent of alkylation were found to correlate with the autoradiographic distribution of tissue-bound 14C-radioactivity in in-situ preparations of the upper gastrointestinal tract following oral exposure to [14C-methyl]-NMBzA. In mice, systemic administration of NMBzA leads to the development of forestomach and lung tumours; in this species, DNA methylation after intraperitoneal injection of NMBzA is highest in liver, followed by lung and forestomach. Administration of [14C-methyl]-NMBzA to mice in the drinking-water led to very high concentrations of alkylated DNA bases in both oesophagus and forestomach. This finding is in good agreement with carcinogenicity studies, which showed 100% carcinoma incidence at these sites. Autoradiographic studies indicate that in rats and mice the metabolism of NMBzA in the oesophagus is largely restricted to the mucosa, whereas in lung, bioactivation occurs predominantly in the bronchial epithelium. In autoradiographs from liver, tissue-bound radioactivity showed a patchy distribution, with predominant reaction in the centrilobular region. In Mongolian gerbils, methylation of lung DNA by a similar subcutaneous dose of [14C-methyl]-NMBzA was greater than in rats and mice, whereas in the remaining tissues, levels of methylated purines were comparatively low. Chronic subcutaneous administration of NMBzA to gerbils caused no tumour within an observation period of two years.     

DNA fragmentation in some organs of rats and mice treated with cycasin.

Cycasin (methylazoxymethanol-beta-D-glucoside) is carcinogenic in several animal species. It produces a variety of malignant tumours, mainly in the liver of mice, and in the liver, kidney and large intestine in rats. It does not appear to be mutagenic in the Ames test, even in the presence of liver microsome fraction, and it is among those carcinogens (less than 10%) ranked as "false negatives" in this test. The ability of cycasin to damage in vivo liver, kidney, lung and colonic DNA of Wistar rats and C57BL/L mice was investigated by means of alkaline elution technique. Oral single-dose administration of cycasin, in the range of 50-400 mg/kg body weight, produced in the rat a clearly evident dose-dependent DNA fragmentation in the liver, and less marked damage to DNA from kidney and colon mucosa. In mice, the same treatment produced dose-dependent DNA damage only in the liver. DNA repair up to 18 h appeared to be incomplete both in mice and rats. Methylazoxymethanol acetate is considered to be an active form of cycasin. While in vivo methylazoxymethanol acetate caused DNA damage, in vitro it appeared inactive and required metabolic activation, possibly consisting in its hydrolysis by esterase activity, to be able to cause DNA fragmentation.