DNA base adducts in urine andwhite blood cells of cancer patients receiving combination chemotherapies which include N-methyl-N-nitrosourea.

Abstract Urinary 3-methyladenine (3-MeAde) excretion andlymphocyte DNA adduct formation was studied in 15 patients receiving methylnitrosourea (MNU) at several dose levels (250 mg, 300 mg and600 mg total dose, 143-385 mg m(-2)) as part of various combination chemotherapies for advanced tumours (malignant melanoma, lymphoblastic lymphosarcorna andHodgkin's disease). Urinary 3-MeAde levels were significantly increased over background in patients at all dose levels (p < 0.001) andthe increases were dose-dependent (r = 0.77, p < 0.01). There were large interindividual variations in the excretion of 3-MeAde at each dose of MNU. In a subset of patients, N7-methyl-2-deoxyguanosine (7-MedG) andO(6)-methyl-2'-deoxyguanosine (O(6)-WedG) levels in DNA from blood leucocytes showed dose-dependent increases, however there were no simple relationships between urinary methylated DNA bases andleucocyte DNA adducts. Levels of adducts in leucocyte DNA (7-MedG, < 17-217 µmol mol(-1) dG; O(6)-WedG, < 1.6-35 µmol mol(-1) dG) were comparable with those reported for other methylating chemotherapeutic drugs. Leucocyte DNA andurinary methyl adducts may be useful markers of individual responses to treatment with methylating drugs.

Individual values of excretion of benzo[a]pyrene metabolites and susceptibility to its carcinogenic effect in rats.

In white outbred LIO rats exposed to multiple intraperitoneal (i.p.) doses (10 mg/kg) of benzo[a]pyrene (BP) in the form of a water-lipid emulsion, individual peculiarities of the excretion of its metabolites, BP-7,8-diol and 3-hydroxy-BP (3-OH-BP) in urine and feces were detected and compared with the carcinogenic effect. Parameters of BP metabolite excretion differed from those found in our previous experiments with rats exposed to single high i.p. doses of BP (100 and 200 mg/kg), dissolved in sunflower oil [11,12]. In comparison with our previous observation, in the present study, the carcinogenic effect was considerably weaker (5/22 versus 10/19). The rats that developed tumours of internal tissues (four peritoneal malignant histiocytomas and one lung lymphosarcoma), excreted higher quantities of BP-7,8-diol in the urine than other rats. The possible implication of monitoring excretion of BP metabolites for predicting individual susceptibility to its carcinogenic effect is discussed.

Alkylation and oxidative-DNA damage repair activity in blood leukocytes of smokers and non-smokers.

The levels of 3 DNA repair enzymes involved in alkylation and oxidative DNA damage repair in human peripheral blood leukocytes were measured in 20 smokers and 17 non-smokers. No differences in O6-alkylguanine-DNA-alkyltransferase (AGT) activity were found between the 2 groups and the AGT distribution within the population appeared to be unimodal. In contrast, the mean activities of both the methylpurine (MeP)- and the 2-6-diamino-4-hydroxy-5N formamidopyrimidine (FaPy)-DNA glycosylases were higher in the smokers, although only the difference between the MeP-DNA glycosylase means was statistically significant. The standard deviations of these 2 enzymes were also higher in the smokers. The MeP-DNA glycosylase activity showed a bimodal distribution when all subjects were considered. This may in part be due to the smoking habit; 83% of the subjects with enzyme activities higher than 500 fmoles/mg protein were current smokers, whilst 85% of the non-smokers had lower enzyme activities. However, if the smokers were considered separately, a bimodal distribution of this enzyme activity could still be observed. No strong correlation was observed between enzyme activity and age, although the slopes of the regression lines of enzyme activity on age were all negative. The relationship between enzyme activities was studied by bivariate distribution and a strong correlation was only found between the MeP-DNA glycosylase and the FaPy-glycosylase, with the highest values of both enzyme activities being observed in the smokers and the lowest in the non-smokers. Our results suggest that the activity of certain DNA repair enzymes can be modulated by environmental exposure.

Biomarkers of individual susceptibility to carcinogens: application for biological monitoring.

In order to develop new markers of individual susceptibility to various human carcinogens, we studied some parameters of formation and metabolism of carcinogens, as well as DNA adducts formation and DNA repair in animals and humans. Following an i.p. administration of benzo(a)pyrene (BP) to the rats, levels of urinary excretion of BP-7,8-diol correlated with tumour latency. A high correlation was found between excretion of this metabolite and BP-DNA adducts level in the liver. Healthy smokers excreted higher quantities of BP-7,8-diol, than smoking lung cancer patients, thus confirming the suggestion on existence of cancer-prone phenotype. N-nitroso compounds formed most efficiently in stomach juice of children with superficial gastritis who therefore could be at high risk of stomach cancer. N-ethyl-N'-nitro-N-nitrosoguanidine induced stomach cancer earlier in monkeys with a low level of DNA repair enzyme, O6-alkylguanine-DNA alkyltransferase (AGT) in gastric mucosa. Overall, these markers can be helpful in predicting individual susceptibility to carcinogens.

Individual levels of activity of O6-alkylguanine-DNA alkyltransferase in monkey gastric mucosa during chronic exposure to a gastrocarcinogen N-ethyl-N'-nitro-N-nitrosoguanidine.

The activity of a DNA repair enzyme, O6-alkylguanine-DNA alkyltransferase (AGT), was studied in gastric mucosa of 15 Macaca fascicularis monkeys before and during chronic oral exposure to the ethylating carcinogen N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) in order to investigate possible causes of inter-individual differences in susceptibility to its gastrocarcinogenic effect. A wide range of AGT activity (307-1903 fmol/mg protein, mean 695) was found before treatment and it decreased during the first year of exposure (means 627, 479 and 452 fmol/mg protein respectively at 6, 12 and 18 months after the beginning of the experiment). The carcinogenesis study is under way and to date four monkeys with low initial AGT level in gastric mucosa died of gastric cancer. The relevance of AGT level measurement for prediction of individual susceptibility to ENNG is discussed.

Biomarkers for individual susceptibility to carcinogenic agents: excretion and carcinogenic risk of benzo[a]pyrene metabolites.

In rats exposed to a single intraperitoneal dose of 200 mg/kg of the environmental carcinogen benzo[a]pyrene (BP) in sunflower oil, significant individual variations in excretion of the BP activation (BP-7,8-diol) and deactivation (3-OH-BP) derivatives were found. Most rats developed peritoneal sarcomas. Only the levels of BP-7,8-diol excreted in the urine correlated directly with the latency of tumor formation. After a similar exposure to a dose of 100 mg/kg BP, Macaca fascicularis monkeys excreted smaller quantities than rats of both metabolites. After rats were given 10 intraperitoneal injections each of 10 mg/kg of BP in a water-lipid emulsion, the excreted levels of both metabolites after the first, fifth, and tenth injection were lower than those of the rats that received 200 mg/kg. BP metabolites were also detected in the urine of lung cancer patients who were heavy smokers. The applicability of monitoring the excretion of the BP metabolites to predicting individual cancer risk is discussed.

O6-alkylguanine-DNA alkyltransferase activity in epidermal tumor and normal epidermal cells of mice of various stocks and strains.

The level of the DNA repair enzyme O6-alkylguanine-DNA alkyltransferase (MGMT) was examined in benign and malignant skin tumors induced with different initiating and promoting agents and from both SENCAR and Sensitive SENCAR Inbred (SSIN) mice. The MGMT levels in the tumors were approximately one-half the level observed in normal surrounding epidermis and in keratinocytes from untreated controls. In addition, a carcinoma-producing cell line, VT 17DT, derived from papillomas in SENCAR mice had no detectable MGMT activity (Mer- phenotype), whereas in the non-tumor forming line, 3PC, MGMT activity was comparable to that in papillomas. The comparatively low level of MGMT in papillomas may contribute to their ease of conversion to squamous cell carcinomas by N-ethyl-N-nitrosourea or n-methyl-N'-nitro-N-nitrosoguanidine. MGMT activity was also determined in the epidermis of non-exposed mice of various stocks and strains. Epidermal MGMT activity was similar to levels in the corresponding livers and was, in general, parallel with stock/strain susceptibility to tumor formation. This is the first report that examined MGMT activity in skin tumors and normal keratinocytes in the mice of several stocks and strains.

Methylation of thymine and uracil with free methyl cations formed due to beta-decay of tritiated methane: possible implication in mutagenesis and carcinogenesis.

Exposure of solid thymine and uracil at room temperature to free methyl cations, produced due to beta-decay of tritiated methane, resulted in formation of their 1-, O2-, 3-, O4-, and 6-methyl derivatives. In addition, uracil formed a 5-methyl derivative (thymine); tritium-containing thymine and uracil were also detected. Both thymine and uracil formed predominantly unidentified products which resulted presumably from their oligomerization. Incubation at -195 degrees C did not markedly change the pattern of reaction products. Aqueous-ammonia solutions of these pyrimidines formed methylated derivatives and considerable amounts of methanol and tritiated water. The possible implication of these reactions in mutagenic and carcinogenic effects of tritium-substituted hydrocarbons is discussed.

5-Bromodeoxyuridine-induced carcinogenesis and its modification by persistent estrus syndrome, unilateral nephrectomy, and X-irradiation in rats.

We showed earlier that 5-bromodeoxyuridine (BrdUrd), which can substitute for thymidine during DNA synthesis, inducing transition mutations, is incorporated into DNA of various tissues when administered to newborn rats and is not subjected to repair processes. The main purpose of the present experiment is to verify if a direct perturbation of DNA would be sufficient to initiate carcinogenesis. Rats aged 1, 3, 7, and 21 days were given BrdUrd s.c. at a dose of 3.2 mg/animal. At 2 months some of the females were subjected to treatment known to induce persistent estrus; at 1 month a group of males underwent removal of one kidney, and groups of males and females were exposed to a single total-body X-irradiation at a dose of 1.5 Gy (150 rads) per rat. In females, treatment with BrdUrd induced tumors of the ovaries, polyps in the uterus, and tumors of the soft tissues, nervous system, forestomach, glandular stomach, and salivary gland; no such tumor occurred in control females. Induction of persistent estrus increased the incidences of ovarian tumors and of malignant tumors of the uterus. Treatment with BrdUrd also increased the carcinogenic effect of X-rays on the mammary gland. In males, BrdUrd induced tumors of the testis (seminomas) and adenomas of the thyroid gland; solitary tumors of the kidney, nervous system, soft tissues, and bladder were also found. Unilateral nephrectomy reduced the incidences of tumors in the testis and pituitary gland, whereas subsequent treatment with X-rays did not alter the incidences of tumors induced by BrdUrd. These studies demonstrated for the first time that a nucleoside analogue, BrdUrd, has carcinogenic potential. Possible molecular mechanisms for its carcinogenicity and for the effects of the promoting factors are discussed.

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 rats received a single i.p. dose (13 mg/kg) of N-[14C]methyl-Nacetoxymethylnitrosamine [( 14C]DMN-OAc), which, under these conditions, selectively induces intestinal tumours. Complete DMN-OAc breakdown occurred within 30 min in both young and old rats but exhalation of 14CO2 continued for over 1 h in young rats and over 3 h in old rats. The highest level of methylation in both young and old rats was found in the DNA of epithelial cells of the colon and in other adjacent abdominal organs (liver and uterus). The initial capacity for excision of the O6-methylguanine from liver DNA was greater in young animals, but further this DNA adduct was repaired more efficiently by the liver of old rats. In the DNA of ileal and colonic enterocytes, O6-methylguanine excision was higher in young than in old animals. The DNA tertiary structure, measured by the sedimentation pattern of nucleoids in neutral sucrose gradient, was damaged in old rats, and, to a lesser extent, in young rats. The non-uniform pattern of DNA damage in young and old animals may be associated with differing carcinogenic effects of DMN-OAc in rats of different ages, a hypothesis which is currently under test.

Incorporation and persistence of 5-bromodeoxyuridine in newborn rat tissue DNA.

One-day-old BDVI rats were given a mixture 5-[3H]bromodeoxyuridine (BrUdR) and [14C]thymidine (each at 20 mg/kg; 12.8 and 3.53 mCi/mmol, respectively) by intraperitoneal injection. DNA was isolated by a phenol procedure from various pooled tissues up to 21 days later and the levels of BrUdR and thymidine incorporation were determined after formic acid hydrolysis and Dowex-50 chromatography. Incorporation increased to a maximum at 12 h then decreased, the decrease of both products occurring in parallel in all tissues examined but differing in kinetics from tissue to tissue. After 12 h, the relative amounts of 3H and 14C showed no consistent or marked decreases and this suggests that BrUdR is not actively removed from DNA by a repair process under these experimental conditions. This was also the case after a much lower dose of these agents (9 micrograms/kg [3H]BrUdR 22.6 Ci/mmol; 1.3 mg/kg [14C]thymidine 53 mCi/mmol) when the 3H/14C ratios in DNA from various tissues were increased relative to the higher dose and showed only slight differences between 15 h and 21 days after administration.

Persistence of methylated purines in the DNA of various rat fetal and maternal tissues and carcinogenesis in the offspring following a single transplacental dose of N-methyl-N-nitrosourea.

Formation and loss of methylated purines in DNA of various fetal and maternal tissues were measured up to 7 days following intravenous administration of N-[14C]methyl-N-nitrosourea to rats on the 21st day of gestation. Methylation products were detected in all tissues examined, the level in maternal liver being higher than in other tissues. The concentrations of 7-methylguanine and 3-methyladenine decreased faster in fetal than in corresponding maternal tissues, due to a higher rate of DNA synthesis in fetal tissues, as determined by incorporation of labelled thymidine. Removal of the promutagenic DNA lesion O6-methylguanine was most efficient in maternal and fetal liver; but it was very poorly repaired in kidney and brain. The persistence of O6-methylguanine relative to 7-methylguanine was highest in the DNA of fetal brain. The principal targets for the transplacental carcinogenic effect of N-methyl-N-nitrosourea under these experimental conditions were fetal neurogenic tissue and kidney; and malignant tumors developed at these sites in 31-34% and 15-16% of male and female descendants, respectively. These results support the concept that a complex interaction between DNA alkylation, repair and replication is the molecular basis of initiation of carcinogenesis by alkylating agents.

Carcinogenicity of single doses of N-nitroso-N-methylurea and N-nitroso-N-ethylurea in Syrian golden hamsters and the persistence of alkylated purines in the DNA of various tissues.

The carcinogenicity of N-nitroso-N-methylurea (NMU) and N-nitroso-N-ethylurea (NEU) has been determined in adult male Syrian golden hamsters following a single i.p. injection or two-thirds of the acute 50% lethal dose, or 30 and 60 mg/kg, respectively. The principal site of action of these agents was the forestomach, squamous cell papillomas of this organ developing in 53 and 61% of the animals receiving the higher doses of NMU and N-nitroso-N-ethylurea, respectively. NMU also induced a low incidence of liver tumors (17%). Very few tumors were seen at other sites. The formation and removal of alkylated purines in DNA was measured in various tissues up to 50 hr after administration of [14C]NMU. Methylation products were detected in all tissues examined, the level in liver being somewhat higher than in other tissues. The removal of 7-methylguanine and 3-methyladenine from DNA occurred at approximately similar rates in all tissues examined, indicating no substantial differences in N-glycosylase activities. Removal of the promutagenic DNA lesion O6-methylguanine varied considerably from tissue to tissue; very little occurred in brain or kidney, while up to 36 and 32% were lost from DNA of intestine and testes, respectively. In the liver, there were relatively small changes in O6-methylguanine levels up to 24 hr; but by 50 hr, 38% had been removed. The persistence of O6-methylguanine relative to 7-methylguanine was highest in the DNA of the brain and intestine and lowest in that of the liver. These results indicate that in this experimental system, the formation and persistence of O6-methylguanine in DNA is insufficient alone to account for the organotropic effect of NMU.

Increased cancer incidence in the progeny of male rats exposed to ethylnitrosourea before mating.

Results from previous experiments have indicated tha persistence of an increased cancer risk in subsequent generations following prenatal exposure to a chemical carcinogen. In the present experiment, the possible role of prezygotic events in determined cancer risk was investigated in the progeny of male rats treated with ethylnitrosourea (ENU) before mating with untreated females. Eight BDVI male rats were given a single i.p. dose of 80 mg/kg bw ENU and each rat was then caged at weeks 1, 2, 3 and 4 after treatment with three untreated females. Fertility was lower and preweaning mortality higher in the experimental group, as compared to controls, particularly at the 4th-week mating. Survival rates after weaning were similar in the progeny of treated males and controls, as was the total incidence of tumours. However, analysis of tumour incidence at the various organ sites showed an increased incidence of neurogenic tumours in the progeny of ENU-treated males, as compared to that of controls.

Incorporation of 5-bromodeoxyuridine into DNA in newborn rat tissues.

The incorporation of bromodeoxyuridine (BUdR) into newborn rat tissue DNA has been determined after i.p. injection of 5-bromo-2'-deoxy-[6-3H]uridine. Incorporation of the unchanged nucleoside was shown by hydrolysis and ion exchange chromatography of extracted DNA. In all tissues examined, more than 90% of the radioactivity incorporated was in the form of bromodeoxyuridine.

In vivo alkylation of foetal, maternal and normal rat tissue nucleic acids by 3-methyl-1-phenyltriazene.

The carcinogen 3-methyl-1-phenyltriazene (MPT) was administered subcutaneously to normal or pregnant BD VI rats and DNA and RNA were isolated from various tissues after 8 h or 15 h, respectively. Sephadex G-10 chromatography of DNA hydrolysates showed the presence of 7-methylguanine in all tissues examined including that of the brain, one of the target organs for tumour induction. The amounts of the minor product, O6-methylguanine, were characteristic of an SN1 reaction mechanism. Dowex-50 chromatography of RNA hydrolysates showed the presence of 7-methylguanine and of the minor product, 3-methylcytosine. The relative amounts, both of the methylated bases in the individual nucleic acids and of 7-methylguanine in DNA and RNA, were similar to those found previously after administration of 3,3-dimethyl-1-phenyltriazene (DMPT). This suggests the involvment of a common alkylating intermediate. De novo incorporation of radioactivity into purine bases was detected in both DNA and RNA although the levels were not related to the amounts of methylation. The results show that MPT is sufficiently stable to alkylate nucleic acids in vivo and are consistent with the hypothesis that this reaction is a prerequisite for tumour induction. Futhermore, they support the proposal that MPT is the active intermediate in the induction of tumours by DMPT.