Metabolic fate of nitrosoproline in the rat.

The metabolism of [U14C]nitrosoproline and [carboxyl-14C]-nitrosoproline was studied in the rat. In most experiments, less than 1% of the administered radioactivity appeared as radioactive CO2 in the expired air, and urinary excretion of radioactivity was rapid and almost complete as the unchanged compound, which was the only radioactive component detected in the urine. The absence of any radioactive urinary components corresponding to proline or its metabolites indicated that no detectable metabolic denitrosation had occurred. Studies of the disappearance of nitrosoproline from the circulating plasma indicated an apparent volume of distribution of 52% of the total body weight, suggesting some penetration by the compound into the intracellular space. There was no significantly detectable covalent binding of radioactivity from the labeled nitrosoproline to DNA ro RNA of the liver and only an extremely low level of binding to liver protein. Taken with the reported noncarcinogenicity of nitrosoproline in rodents, the above findings suggest that proline may be a suitable nitrosatable substrate for use in tests of endogenous nitrosation reactions in animals and human subjects.

Alpha-difluoromethylornithine inhibits N-nitrosomethylbenzylamine-induced esophageal carcinogenesis in zinc-deficient rats: effects on esophageal cell proliferation and apoptosis.

Sustained, increased cell proliferation induced by dietary zinc deficiency in rats plays a critical role in esophageal carcinogenesis. It is the determining factor that converts an otherwise nontumorigenic dose of N-nitrosomethylbenzylamine (NMBA) into a highly tumorigenic one. We studied whether the increased esophageal cell proliferation and susceptibility to NMBA-induced carcinogenesis induced by zinc deficiency can be inhibited by alpha-difluoromethylornithine (DFMO), an enzyme-activated, irreversible inhibitor of ornithine decarboxylase (the first enzyme in polyamine synthesis). Weanling rats were divided into four groups: Zn+/DFMO-, Zn+/DFMO+, Zn-/DFMO-, and Zn-/DFMO+. They were fed ad libitum either a zinc-sufficient (Zn+, 75 ppm zinc) or a zinc-deficient (Zn-, 4 ppm zinc) diet and given either deionized water (DFMO-) or 1% DFMO in deionized water (DFMO+). After 5 weeks, 5-19 animals from each group were sacrificed after in vivo 5-bromo-2'-deoxyuridine labeling to detect cells in S phase. The remaining animals in each group were given a single intragastric dose of NMBA at 2 mg/kg and sacrificed 12 weeks later for tumor incidence analysis. At week 5, DFMO treatment greatly decreased (by 48-82%) the levels of putrescine and spermidine in rat esophagus, colon, and liver, irrespective of dietary zinc intake. The increased esophageal cell proliferation induced by dietary zinc deficiency, as measured by the labeling index, the number of labeled cells, and the total number of cells, was substantially reduced by DFMO. This was accompanied by an increase in the rate of apoptosis. In addition, the expression of bax protein, an apoptosis accelerator, was markedly stronger in esophagi from Zn-/DFMO+ animals that showed increased apoptosis, whereas increased expression of bcl-2, an inhibitor of apoptosis, was only seen in the highly proliferative, zinc-deficient esophagus (Zn-/DFMO-). At week 12 after NMBA dosing, DFMO reduced the incidence of esophageal tumors from 80 to 4% in zinc-deficient rats. Our data showed that DFMO effectively inhibited the increased esophageal cell proliferation induced by dietary zinc deficiency and reduced the incidence of esophageal tumors induced by a single dose of NMBA in zinc-deficient animals. Our results also indicate a role for increased apoptosis in the mechanism(s) whereby DFMO brings about the inhibition of cell proliferation and tumor induction. These findings support a role for DFMO as a chemopreventive agent.

Measurement of 7-methylguanine as an estimate of the amount of dimethylnitrosamine formed following administration of aminopyrine and nitrite to rats.

We have demonstrated that there is a dose-related increase in the excretion of 7-[methyl-14C]methylguanine ( [14C]m7Gua) following p.o. administration of di[methyl-14C]methylnitrosamine to rats. Urine was collected for 24 hr after di[methyl-14C]methylnitrosamine administration, and the purines were precipitated from an aliquot of the urine with silver nitrate. Purines were released from the precipitate with HCl, and [14C]m7Gua was quantified by chromatography on an Aminex A-6 column. The excretion of [14C]m7Gua increased linearly with the dose of dimethylnitrosamine. This relationship was used to estimate the amount of di[methyl-14C]methylnitrosamine formed in the reaction of [14C]aminopyrine with sodium nitrite in rats gavaged with these compounds. The dose of dimethylnitrosamine was also estimated from the amount of alkylation of liver DNA in the same animals. These estimates usually differed by less than a factor of 2. [14C]aminopyrine and sodium nitrite were administered. The possibility of using this assay to obtain data on nitrosation in humans is discussed.

Early deregulation of the the p16ink4a-cyclin D1/cyclin-dependent kinase 4-retinoblastoma pathway in cell proliferation-driven esophageal tumorigenesis in zinc-deficient rats.

The p16ink4a-cyclin D1/cyclin-dependent kinase 4 (Cdk4)-retinoblastoma (Rb) pathway has emerged as a critical target in oncogenesis. The zinc-deficient (ZD), N-nitrosomethylbenzylamine (NMBA)-induced rat esophageal cancer model provides a tool to study cell proliferation and cell cycle control in cancer initiation. Weanling rats were fed a ZD or zinc-sufficient (ZS) diet for 5 weeks, and then given a dose of NMBA. After 14 weeks, esophageal tumor incidence was 88% in ZD rats with highly proliferative esophagi versus 0% in ZS rats. Expression of p16ink4a, cyclin D1, Cdk4, and Rb in relation to that of proliferating cell nuclear antigen was characterized in esophagi by immunohistochemistry at 0, 24, and 48 h, and 1, 3, 7, 10, and 14 weeks after NMBA treatment. As early as 24 h, proliferating cell nuclear antigen-positive focal hyperplastic lesions were detected in the suprabasal layers of ZD esophagi. At the same time, overexpression of cyclin D1, Cdk4, and Rb was found in the corresponding lesion in adjacent esophageal sections. By contrast, p16ink4a expression was reduced or absent. At all time points, p16ink4a showed reduced nuclear staining in ZD esophagi compared with that in ZS esophagi. In addition, increased expression of the hyperphosphorylated forms of Rb was detected in ZD esophagi by immunoblotting. Importantly, tumors were consistently observed in ZD esophagi at very early time points. These data, obtained using a unique in vivo model for esophageal cancer with rapid tumor induction, provide strong evidence for a link between deregulation of the p16ink4a-cyclin D1/Cdk4-Rb pathway and the initiation of esophageal tumors.

Interspecies scaling of the pharmacokinetics of N-nitrosodimethylamine.

The pharmacokinetics of N-nitrosodimethylamine was studied in patas monkeys following i.v. doses of 0.5, 1.0, and 5.0 mg/kg and a p.o. dose of 1.0 mg/kg, and in Swiss mice at i.v. doses of 1.0 and 2.0 mg/kg. In the patas monkey the pharmacokinetics was linear over the i.v. dose range studied. The mean clearance (Cl), steady-state volume of distribution (Vss), mean residence time, and elimination half-life (t 1/2) were 103.3 +/- 26.7 (SD) ml/min, 3061 +/- 821 ml, 30.8 +/- 10.8 min, and 21.1 +/- 8.5 min, respectively. Assuming that the pharmacokinetics was linear at the p.o. dose used, the p.o. bioavailability of N-nitrosodimethylamine in the monkey was 49%. The pharmacokinetics was also linear in mice, and the average Cl, Vss, mean residence time, and t 1/2 were 3.81 ml/min, 21.0 ml, 5.5 min, and 11.9 min, respectively. These data and data for rats, hamsters, rabbits, dogs, and pigs taken from the literature were used to scale Cl and Vss to body weight using the allometric equation. The resulting equation for Cl was Cl = 49.7B0.998 and the equation for Vss was Vss = 748B1.05 where B is body weight in kg. The fit of the data to the equation was excellent in both cases. Using these equations and assuming a body weight of 70 kg for humans, the Cl and Vss for N-nitrosodimethylamine in humans are estimated to be 3450 ml/min and 64,800 ml, respectively.

Reduced blood clearance and increased urinary excretion of N-nitrosodimethylamine in patas monkeys exposed to ethanol or isopropyl alcohol.

Low concentrations of N-nitrosodimethylamine are metabolized in rodent and human liver by cytochrome P450IIE1, an activity competitively inhibitable by ethanol. In rodents coadministration of ethanol with N-nitrosodimethylamine results in increased tumorigenicity in extrahepatic organs, probably as a result of reduced hepatic clearance. To test this concept in a primate, the effects of ethanol cotreatment on the pharmacokinetics of N-nitrosodimethylamine were measured in male patas monkeys. Ethanol, 1.2 g/kg given p.o. before i.v. N-nitrosodimethylamine (1 mg/kg) or concurrently with an intragastric dose resulted in a 10-50-fold increase in the area under the blood concentration versus time curves and a 4-13-fold increase in mean residence times for N-nitrosodimethylamine. Isopropyl alcohol, 3.2 g/kg 24 h before N-nitrosodimethylamine, also increased these parameters 7-10-fold; this effect was associated with persistence of isopropyl alcohol and its metabolic product acetone, both IIE1 inhibitors, in the blood. While no N-nitrosodimethylamine was detected in expired air, trace amounts were found in urine. Ethanol and isopropyl alcohol pretreatment increased the maximum urinary N-nitrosodimethylamine concentration 15-50-fold and the percentage of the dose excreted in the urine by 100-800-fold. Thus ethanol and isopropyl alcohol greatly increase systemic exposure of extrahepatic organs to N-nitrosodimethylamine in a primate.

Deregulation and overexpression of c-fos proto-oncogene in rat renal cell-lines and primary tumors induced by dimethylnitrosamine.

Rat renal mesenchymal tumors induced by the chemical carcinogen N-dimethylnitrosamine (DMN) and cell-lines derived from kidneys of rats after DMN treatment were found to express abnormal steady state levels of c-fos RNA. This overexpression was not found to arise by gene amplification or rearrangement, but the c-fos gene appeared to be deregulated resulting in increased transcription. The consistent nature of this observation suggests a function for c-fos gene overexpression in tumorigenesis of the rat kidney by DMN.

Alpha-difluoromethylornithine induction of apoptosis: a mechanism which reverses pre-established cell proliferation and cancer initiation in esophageal carcinogenesis in zinc-deficient rats.

Alpha-difluoromethylornithine (DFMO) is an irreversible inhibitor of ornithine decarboxylase, the first enzyme in polyamine synthesis. Previous work showed simultaneous administration of DFMO and a zinc-deficient (ZD) diet to weanling rats from the beginning inhibited the onset of zinc-deficiency-induced esophageal cell proliferation by activating apoptosis and reduced the incidence of N-nitrosomethylbenzylamine (NMBA)-induced esophageal cancer. Because esophageal cancer initiation by NMBA is very rapid in ZD rats, this study determined whether DFMO is effective in preventing esophageal carcinogenesis when administered after the establishment of a carcinogenic environment. Weanling rats were given a ZD diet for 5 weeks to establish sustained increased esophageal cell proliferation and then an intragastric dose of NMBA. Thereafter, 20 rats were switched to DFMO-containing water while nine control ZD animals remained on deionized water; all of the animals continued on the ZD diet. Esophagi were collected 15 weeks later. The upper portion was processed for immunohistochemical analysis of cell proliferation, apoptosis, and expression of related genes, and the lower was processed for polyamine content. DFMO substantially reduces the levels of esophageal putrescine and spermidine and esophageal tumor incidence from 89 to 10% in ZD rats. Importantly, DFMO-treated ZD esophagi display increased rate of apoptosis accompanied by intense bax expression and greatly reduced cell proliferation by proliferating cell nuclear antigen expression. In addition, the p16(ink4a)/retinoblastoma control at G1 to S, deregulated in ZD esophagi, is restored after DFMO treatment. These results demonstrate that DFMO, a highly effective chemopreventive agent in esophageal carcinogenesis, reverses and counteracts esophageal cell proliferation/cancer initiation in ZD animals by way of stimulating apoptosis.

Dietary zinc deficiency enhances esophageal cell proliferation and N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumor incidence in C57BL/6 mouse.

The effect of zinc deficiency on N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumor formation in rats has been well documented. Our previous work showed that zinc deficiency and its associated increased esophageal cell proliferation were of paramount importance in esophageal tumor development in the NMBA-rat model. However, there has been no report concerning zinc deficiency and NMBA-induced esophageal tumor formation in mice. In this study, weanling C57BL/6 mice were fed ad libitum with either a zinc-sufficient or a zinc-deficient diet containing 3-4 ppm of zinc, and received six intragastric doses of NMBA (2 mg/kg; twice weekly for 3 weeks). The animals were sacrificed 46 weeks later after in vivo bromodeoxyuridine (BrDU) labeling followed by immunohistochemical detection of cells in S-phase. At 46 weeks, the tumor incidences in zinc-deficient mice were 57, 100, and 100% respectively, in the esophagus, forestomach and squamocolumnar junction with the glandular stomach (SCJ), as compared to 17, 39, and 67% in the corresponding tissue of zinc-sufficient mice. The difference between the two dietary groups was significant at P < 0.02 for the esophagus, and P < 0.001 for the forestomach and the SCJ. BrDU labeling revealed that the esophageal labeling index and the number of labeled cells were increased by zinc deficiency. These results support a role of increased cell proliferation in esophageal carcinogenesis in the mouse.

Protein bound carboxyl-methyl ester as a precursor of methanol formation during oxidation of dimethylnitrosamine in vitro.

Protein modification with dimethylnitrosamine was studied in vitro in the presence of hamster liver microsomal fraction. Incorporation of radioactive methyl groups from dimethylnitrosamine into the exogenously added protein was dependent on the microsomal mixed function oxidase system. The methylation yielded chemically labile and stable products. The former was completely hydrolyzed by the mild alkaline treatment, pH 7.4, 100 degrees C, for 5 min and the hydrolytic product was identified as methanol indicating that the activated methyl groups from dimethylnitrosamine were incorporated into a protein as a carboxyl-methyl ester. Thus, it is suggested that methanol, recovered as one of the products during the biodegradation of dimethylnitrosamine [8], is derived, at least in part, from protein carboxyl-methyl ester which is unstable under physiological conditions.

Factors influencing the activity of dimethylnitrosamine demethylase in hamster, rat and chicken liver microsomes.

In vitro metabolism of dimethylnitrosamine (DMN) by liver microsomal fractions of hamster, rat and chicken revealed that the three species under certain assay conditions, were capable of metabolizing DMN at different rates (hamster greater than rat greater than chicken). The magnitude of the demethylase activity was found to be dependent on the nature of the buffer, the concentration of cytochrome P-450 (P-450) and the concentration of the substrate DMN. Enzyme activity was higher in Hepes buffer than in the phosphate buffer. Concentrations of phosphate higher than 20 mM inhibited the activity of the rat and chicken enzymes. This effect of phosphate was not a consequence of increase in ionic strength since KCl over a wide range of concentration failed to inhibit the activity.

Differential effects on the metabolism of dimethylnitrosamine and aflatoxin B1 by hepatic microsomes from senescent rats.

The ability of hepatic microsomes from senescent rats to metabolize the two potent hepatocarcinogens dimethylnitrosamine (DMN) and aflatoxin B1 (AFB1) was investigated. Seven and 24-month-old male Sprague-Dawley rats were used. Liver weights, and microsomal protein per gram tissue weight were higher, whereas cytochrome P-450 and cytochrome b5 were significantly lower in older rats. Glutathione S-transferases and NADPH cytochrome c reductase activities were dramatically reduced in senescent rats. There was no difference in the formation of formaldehyde from DMN in vitro (31 vs. 34 pmol/nmol P-450) between the young and old rats. In contrast, increased microsome mediated binding of AFB1 to DNA was observed in older rats (116 vs. 228 pmol/nmol P-450) suggesting the possibility of either quantitative or qualitative changes in P-450 species. Additionally the cytoplasmic GSH S-transferases from older rats affected lower inhibition of binding of AFB1 to DNA. These results indicated differential abilities in the hepatic microsomal metabolism of these two carcinogens which may cause differential effects of these carcinogens in senescent rats.

Effect of purified rat and hamster hepatic glutathione S-transferases on the microsome mediated binding of aflatoxin B1 to DNA.

Rat and hamster liver cytosolic glutathione (GSH) S-transferases purified by GSH-affinity chromatography have been examined for their effects on the microsome mediated binding of aflatoxin B1 (AFB1) to DNA and on the conjugation of AFB1-2,3-epoxide with GSH. Like previous studies with cytosolic preparations (Raj et al. (1984) Carcinogenesis 5, 879), our present study with purified GSH S-transferases showed 2-3-fold more inhibitory activity of AFB1-DNA binding with hamster than that with the rat. Concomitant with the inhibition of AFB1-DNA binding, increase in AFB1-GSH conjugation occurred. Subunit compositions of GSH S-transferases indicate preponderance of Yb and Ya subunits in the hamster and rat, respectively. The role of GSH S-transferases in modulating AFB1-DNA binding and AFB1 induced hepatocarcinogenesis is discussed.

Alkylation of protein by methyl methanesulfonate and 1-methyl-1-nitrosourea in vitro.

Methylation of horse heart cytochrome c has been examined in vitro with [methyl-14C]methanesulfonate (MMS) and [1-methyl-14C]-1-nitrosourea (MNU) as alkylating agents. Analysis of protein hydrolyzates by an automatic amino acid analyzer indicates that, at pH 9.0 with MMS, epsilon-N-monomethyl-lysine is found to be the only major methylated basic amino acid. On the other hand, the identity of the predominant basic amino acid residue which is [methyl-14C]-labeled by MNU cannot be determined at present. Peptide mapping of chymotryptic digests of cytochrome c after reaction with MMS reveals a lack of specificity in methylation of a specific lysine residue in this hemoprotein.

Effect of glutathione modulation using buthionine sulfoximine on DNA methylation by dimethylnitrosamine in the rat.

An in vivo study was carried out in order to determine whether glutathione (GSH) might serve as a scavenger for the supposed electrophilic methylating fragment derived from dimethylnitrosamine (DMN) and thus function to decrease the degree of cellular macromolecule interaction, estimated by measuring the DNA methylation yield. After a 4-hr pretreatment with DL-buthionine-SR-sulfoximine (BSO), a specific inhibitor of GSH synthesis, male Sprague-Dawley rats were dosed with radiolabeled DMN (250 micrograms/kg). Four hours later the animals were killed and the livers and kidneys were excised. The DNA isolated from these organs was hydrolyzed in mild acid, and the liberated purines were quantified utilizing HPLC and liquid scintillation counting. The 70-75% GSH depletion in the liver and kidney resulting from BSO pretreatment did not have any significant effect on the degree of DNA methylation as assessed by the 7-methylguanine/guanine yield. In control experiments we found that DMN doses greater than 1 mg/kg had a marked effect on liver and kidney GSH levels after 4 hr.

Studies on the distribution of O6-methylguanine-DNA methyltransferase in rat.

O6-Methylguanine-DNA methyltransferase, a DNA repair enzyme which transfers the methyl group of O6-methylguanine residue to a cysteinyl residue in the methyltransferase itself, was examined in rat organs by quantifying the S-methylcysteine formed in the methyl acceptor protein. Among the various organs examined, the spleen exhibited the highest enzyme specific activity followed by the thymus, liver, lung and testis. Brain had the lowest activity. The patterns of subcellular distribution of the methyltransferase in spleen and liver were different: while 75-80% of the activity was present in the nuclear fraction of the spleen, 54% of the activity in the liver was found in the nuclei and 35% in the cytosolic fraction. Forty-five and thirty-five percent of the total nuclear enzyme activity could be extracted with 1 M and 2 M NaCl solutions, respectively, indicating that the repair enzyme is not tightly bound to the nuclear matrix. When isolated nuclei were incubated with [methyl-3H]DNA substrate and subsequently fractionated into histone and non-histone protein fractions, over 90% of the radioactivity was coeluted on a Bio-Rex 70 column with the non-histone fraction and only a negligible amount of radioactivity was found to be associated with the histone fraction. The molecular mass of the [methyl-3H]methyltransferase in the non-histone fraction was determined to be 23,000, and its pI value was found to be 6.6 by two-dimensional polyacrylamide gel electrophoresis.

The metabolism of N-nitrosomethylaniline.

The distribution in the body, rate of disappearance from organs, tissues, and blood, and excretion in the urine of N-nitrosomethylaniline (NMA, N-nitrosophenylmethylamine) was investigated after various single doses given IP to rats. The compound was distributed fairly evenly throughout the body with no preferential concentration in the esophagus, its target organ for carcinogenicity. The high lipid solubility of NMA did not lead to any increased accumulation in adipose tissue. According to its rate of disappearance from circulating blood and tissues of rats, and from the whole bodies of mice after injection, NMA appeared to be rapidly metabolized. Methylaniline (MA), the parent amine, was found in the urine after administration of NMA but the amounts present were small relative to the dose of NMA. Administered MA was mainly excreted unchanged in the urine, suggesting that denitrosation of NMA could only be a minor metabolic pathway. No volatile nitrosamines were found in the urine or blood of rats given NMA, indicating that little, if any, transnitrosation could have occurred to yield these compounds.

Effect of food restriction on the metabolism of dimethylnitrosamine (NDMA) in rats.

The influence of food restriction on the macromolecular interactions of the hepatocarcinogen dimethylnitrosamine (NDMA) in the livers of male Sprague Dawley rats was investigated. Two-three month old rats were food restricted (FR) (40% with respect to ad libitum fed rats) for three weeks. The liver weight, total protein, microsomal and cytoplasmic protein, cytochrome P-450 and DNA content per whole liver were all reduced significantly in food restricted rats. Five hours after a single dose of (14C) NDMA (28 mg/k.b.w., 21 microCi/rat) the levels of 7-methylguanine increased in restricted rats by 32%. Cytochrome P-450 mediated generation of HCHO from NDMA (1.8 fold) in restricted rats was greater. Binding of NDMA derived radioactivity to total hepatic proteins decreased by 46% in restricted rats. These results suggest that food restriction enhances the metabolic activation of dimethylnitrosamine in Sprague Dawley rats.

DNA-methylation by nitrosocimetidine and N-methyl-N-nitro-N-nitrosoguanidine in the intact rat.

The ability of the nitroso derivative of the drug cimetidine to interact with cellular macromolecules in the intact rat was investigated. Radio-labelled nitrosocimetidine (NC) was shown to methylate DNA in a variety of tissues in the rat after oral administration. Radioactivity was also detected in the RNA and protein extracted from these same tissues. Methylation of DNA by the parent compound, cimetidine, was not detected in any of the tissues studied. For comparison, the DNA methylation produced by the carcinogen N-methyl-N-nitro-N-nitrosoguanidine (MNNG) dosed orally was measured. DNA alkylation by MNNG was found to be approx. 2-36 times greater than that produced by NC, varying with the tissues studied. The highest yield of DNA alkylation was found in the stomach for MNNG and the small intestine for nitrosocimetidine suggesting pharmacokinetic differences.

Evidence for metabolism of N-nitrosoproline.

The metabolism of nitrosoproline (NPRO) was re-investigated in uni- and bilaterally nephrectomized rats that have reduced or absent ability to excrete urine. About 1% of the administered radioactivity from L-[U-14C]-NPRO appeared as 14CO2 in the expired air and the production of 14CO2 was time-dependent over a period of 23 h. As compared with sham-operation, uni- or bilateral nephrectomy did not significantly increase the amount of NPRO metabolism, though urinary excretion of radioactivity was decreased in the unilaterally nephrectomized animals. In microsome-mediated and in vitro enzyme-free (Udenfriend-hydroxylating) systems covalent binding of [2,3,4,5-3H]NPRO to exogenous calf thymus DNA was demonstrated. The above findings confirm that in vivo metabolism of NPRO is possible, albeit, to a very small extent.