Accumulation of O6-methylguanine in non-target-tissue deoxyribonucleic acid during chronic administration of dimethylnitrosamine

1. BD-IV rats were given labelled dimethylnitrosamine (2 mg/kg) by stomach tube on weekdays (Monday to Friday) for up to 24 weeks. The rats killed after 2, 4, 8, 16 and 24 weeks of treatment (72 h after the final dimethylnitrosamine gavage) and DNA was isolated from the pooled livers, kidneys and lungs. Purine bases were released from the DNA by mild acid hydrolysis and separated by Sephadex G-10 chromatography. 2. Throughout the experiment, the content of 7-methylguanine in liver DNA was approx. 16 times that in kidney and lung. The amount of this product increased in the DNA of all three tissues up to 16 weeks, but by 24 weeks had decreased by 20% in the liver and 46% in the other tissues. 3. O6-Methylguanine was not detected in liver DNA, but was easily measured in kidney and lung DNA after 4 weeks of dimethylnitrosamine administration. The amount of O6-methylguanine in kidney and lung DNA increased relative to that of 7-methylguanine, and by 24 weeks was 60% of the 7-methylguanine content in both tissues. 4. Incorporation of radioactive C1 breakdown products of dimethylnitrosamine into normal purines in DNA increased continuously in all three tissues. 5. The results are discussed with respect to the specific hepatocarcinogenic effect of chronic administration of dimethylnitrosamine and the possible contribution of increased DNA repair and DNA synthesis.     

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-methylguanine in liver DNA. The methyl moiety in these reactions might be derived from the cellular S-adenosylmethionine pool because radioactivity administered to these rats as methionine rapidly appears in the DNA as methylated guanine. An increased incorporation of radioactivity into 5-methylcytosine was previously reported followed by subsequent suppression. This increased radiolabeling of 5-methylcytosine coincided with time of maximal DNA guanine methylation. To determine the nature of S-adenosylmethionine metabolism during the period of DNA methylation induced by hydrazine treatment, and to determine if the increased radiolabeling of 5-methylcytosine at this time reflected an actual increase in 5-methylcytosine synthesis, liver DNA synthesis and S-adenosylmethionine levels and turnover were assayed. Liver S-adenosylmethionine concentrations varied slightly between control rats and hydrazinetreated rats during the first five hours after hydrazine administration, and no difference was detectable in the incorporation of administered [3H]methionine into S-adenosylmethionine. Because S-adenosylmethionine specific radioactivity in hydrazine-treated rats was not different from control rats, the previously observed increased radiolabeling of 5-methylcytosine appeared to represent an actual increase in synthesis. This conclusion was supported by finding that incorporation of radioactive thymidine into DNA was also accelerated immediately following hydrazine administration, again followed by a decrease. 5-Methylcytosine sythesis, therefore, appears to follow DNA synthesis during hydrazine toxicity, and formation of 7-methylguanine and O6-methylguanine in liver DNA of hydrazine-treated rats occurs during a short period of increased DNA sythesis and 5-methylcytosine formation very early in hydrazine toxicity.     

Resuming of thermoregulation in rats during deep hypothermia with EDTA without rewarming the body

Carcinogen-resistant inbred DRH rats developed from the Donryu strain showed a remarkably low incidence of liver tumors when they were fed diets containing hepatocarcinogens such as 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB). In this work, we examined various characteristics of male DRH and Donryu rats during 3'-Me-DAB administration for 8 weeks. 32P-Postlabeling analysis showed that essentially similar levels of DNA-adducts were generated by the metabolites of 3'-Me-DAB in the livers of these two strains of rats at several time points. However, both GADD45 (growth arrest and DNA damage-inducible) and O6-methylguanine methyltransferase (putatively DNA damage-inducible) mRNA levels were increased significantly in Donryu rat livers, but were increased to a lesser extent in DRH rats. [3H]Thymidine incorporation into hepatic DNA began to increase around 10 to 20 days after the start of 3'-Me-DAB administration in Donryu rats probably due to DNA repair, while no significant change occurred in DRH rats under the same conditions. Furthermore, inductions of heme oxygenase (due to degradation of heme-proteins) and hepatocyte growth factor (HGF; cell death and regeneration of hepatocytes) mRNAs were greater in Donryu rat livers than those of DRH, suggesting that the former were more sensitive to cytotoxic effects of 3'-Me-DAB than the latter. Another remarkable difference observed between these two strains was the significant induction of cytochrome P-450 2E1 mRNA in Donryu rat livers; this may contribute to the generation of reactive oxygen intermediates. Finally, increases of glutathione S-transferase (P-form) and gamma-glutamyltranspeptidase mRNAs as marker enzymes of preneoplastic changes of hepatocytes were clearly seen only in Donryu rat livers at 6 to 8 weeks after the start of 3'-Me-DAB administration. These results indicate that the different susceptibility to hepatocarcinogenesis between these two strains of rats may arise from events other than the DNA adduct formation.     

Methylated purines in the deoxyribonucleic acid of various Syrian-golden-hamster tissues after administration of a hepatocarcinogenic dose of dimethylnitrosamine

1. DNA was extracted from livers, kidneys and lungs of Syrian golden hamsters at various times (up to 96h) after injection of a hepatocarcinogenic dose of [14C]dimethylnitrosamine. Purine bases were released from the DNA by mild acid hydrolysis and separated by Sephadex G-10 chromatography. 2. At 7h after dimethylnitrosamine administration liver DNA was alkylated to the greatest extent, followed by that of lung and kidney, the values for which were 8 and 3% respectively of those for liver. 3. The O6-methylguanine/7-methylguanine ratios were initially the same in all three organs and in the liver DNA of rats under similar conditions of dose. 4. O6-Methylguanine was the most persistent alkylated purine in all three hamster tissues. There was evidence for excision of 7-methyl-guanine, the highest activity for this being present in the liver. 5. Detectable amounts of the minor products 3-methyladenine, 1-methyladenine, 3-methylguanine and 7-methyladenine were present in most hamster tissues, and their individual rates of loss from liver DNA were determined. 6. Ring-labelling of the normal purines in DNA was highest in the liver, followed closely by the lung (80% of that in liver) whereas the kidney had very low incorporation (3% of that in liver). 7. The results are discussed with respect to the hepatotoxicity of dimethylnitrosamine, the miscoding potential of the various alkylation products and the induction of liver tumours in hamsters.     

Repair of DNA lesion O6-methylguanine in hepatocellular carcinogenesis

Evidence from both experimental carcinogenesis and studies in human cirrhotic liver suggest that defective repair of the promutagenic DNA base lesion, O6-methylguanine, is a factor in the multistep process of hepatocellular carcinogenesis. Ubiquitous environmental alkylating agents such as N-nitroso compounds can produce O6-methylguanine in cellular DNA. Unrepaired, O6-methylguanine can lead to the formation of G --> A transition mutations, a known mechanism of human oncogene activation and tumour suppressor gene inactivation. Combined treatment of rodents with an agent producing O6-methylguanine in DNA, and an agent promoting cell proliferation, leads to development of hepatic nodules and hepatocellular carcinoma (HCC), cell division, hence DNA replication, being required for the propagation of tumorigenic mutation(s) in hepatocyte DNA. The paramount importance of O6-methylguanine in hepatocellular carcinogenesis is indicated by the observation that transgenic mice engineered to have increased hepatic levels of repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) are significantly less prone to hepatocellular carcinogenesis following alkylating agent treatment. Cirrhosis is a universal risk factor for development of human HCC, and a condition that is characterized by increased hepatocyte proliferation as a result of tissue regeneration. Levels of the human repairing enzyme for O6-methylguanine were found to be significantly lower in cirrhotic liver than in normal tissue. In accord with findings from animal models, this suggested a mechanism in which persistence of O6-methylguanine due to defective DNA repair by MGMT, together with increased hepatocyte proliferation, might lead to specific gene mutation(s) and hepatocellular carcinogenesis. Screening for the presence and persistence of O6-methylguanine in human DNA presently involves formidable technical difficulty. Indications are that such limitations might be overcome by the use of an ultrasensitive method such as immuno-polymerase chain reaction (PCR). This approach should allow parallel measurement of DNA adduct and repair enzyme in routine liver biopsy samples. It might also enable investigation of O6-methylguanine in human genes specifically associated with hepatocellular carcinogenesis. Given the wide variation in human MGMT levels observed between individuals, tissues, and cells, this technology should be adapted to permit the ultrasensitive localisation and measurement of adducts and repairing enzyme in liver biopsy tissue sections. Ability to ultrasensitively measure O6-methylguanine, and its repair enzyme, should prove valuable in the risk assessment of cirrhotic patients for developing hepatocellular carcinoma.     

The role of hepatocyte heterogeneity in the initiation of hepatocarcinogenesis

N-Nitrosodimethylamine (NDMA) is a carcinogen in rat liver while N-nitrosomethylbenzylamine (NMBzA) produces no liver tumors but is a potent esophageal carcinogen in the rat. Both nitrosamines, however, are metabolically activated in the liver and methylate hepatic DNA. The reasons for their different carcinogenic properties in rat liver are unclear. Here we show that as expected, NDMA produces large numbers of putative initiated hepatocytes that overexpress the placental form of glutathione S-transferase (GST-P+ hepatocytes). Hepatocyte division induced by the hepatotoxic effect of NDMA occurs principally in the periportal region of the liver lobule, while O6-methylguanine formation is principally in the DNA of perivenous cells. These two effects lead to the production of GST-P+ hepatocytes in roughly equal numbers throughout the liver lobule. NMBzA also induces the formation of a small, but significant number of GST-P+ hepatocytes. The NMBzA-induced GST-P+ hepatocytes are localized within the perivenous zone of the liver lobule. Since, unlike NDMA, NMBzA produces no hepatocellular necrosis and hence does not induce regenerative cell division, these results suggest that NMBzA initiates only those hepatocytes adjacent to the hepatic vein that are spontaneously dividing at the time their DNA becomes methylated by the nitrosamine. We used partial hepatectomy to stimulate cell division in specific regions of the liver lobule. When the peak of DNA methylation produced by NMBzA in the perivenous cells coincided with a peak of cell division in that region, an increased number of GST-P+ hepatocytes was induced. Our results suggest that the potency of initiating agents in the liver depends both on their ability to form mutagenic lesions in DNA and to induce division in the specific hepatocytes that contain the modified DNA.     

Excision of O6-methylguanine from DNA of various mouse tissues following a single injection of N-methyl-Nitrosourea

The persistence of O6-methylguanine produced by a single dose of N-methyl-N-nitrosourea (MNU) was determined in DNA of various murine tissues and compared with the location of tumours induced by MNU and related alkylating carcinogens in this species. A/J and C3HeB/FeJ mice received a single intravenous injection of MNU (10 mg/kg) and were killed at different time intervals ranging from 4 h to 7 days. The rate rate of loss of O6-methylguanine from brain DNA was considerably slower than from liver DNA; tumours have been found in both organs after administration of MNU and other alkylnitrosoureas. There was no difference in the rate of excision from cerebral DNA of A/J and C3HeB/FeJ mice, although these strains differ significantly in their susceptibility to the neurooncogenic effect of MNU and related carcinogens. Excision of O6-methylguanine from hepatic DNA was significantly slower in A/J than in C3HeB/FeJ mice; both strains habe been found to develop hepatic carcinomas following MNU administration. Seven days after the injection of 3H-MNU, O6-methylguanine concentrations were highest in brain and lung DNA, lowest in the liver, and intermediate in kidney, spleen, small intestine and stomach. The lung is a principal target organ for tumour induction by MNU and other carcinogens in mice; however, neural tumours are usually induced at a low incidence. The results obtained do not contradict the hypothesis that O6-alkylation of guanine in DNA is a critical event in the initiation of tumour induction by alkylating agents. However, the location of tumours produced in mice does not seem to depend solely on the formation and persistence of O6-alkylguanine in DNA.     

Transplantation as a tool to study progenitors within the vertebrate nervous system

The dose responses for several effects of low-level limited exposures to 2-acetylaminofluorene (AAF) in the livers of male Fischer 344 rats were measured and a subsequent phenobarbital tumor promotion regimen was used to manifest initiation of carcinogenesis. Three doses over a 10-fold range yielding cumulative total exposures of 0.126, 0.42, and 1.26 mmol AAF/kg body weight were achieved by daily intragastric instillation for up to 12 weeks with interim terminations. This was followed by 24 weeks administration of 500 ppm phenobarbital (PB) in the diet to promote liver tumor development. At 12 weeks at the end of AAF administration, all exposures produced adducts in liver DNA, measured by 32P postlabeling, and the level of adducts increased with exposure, except that the high exposure did not produce a dose proportional increase. Measurement of arylsulfotransferase activity, a key enzyme in the metabolic activation of AAF, revealed that in livers from the high exposure animals, the enzyme was inhibited. To assess for toxicity, the centrilobular zone of glutamine synthetase-positive hepatocytes was quantified immunohistochemically at 12 weeks. The area of the zone was reduced in the high exposure group and there was a trend to reduction in relationship to exposure. The two lower exposures to AAF produced no increase in cell proliferation, whereas the high exposure resulted in a marked increase, about 8-fold over controls. Initiation was assessed by induction of hepatocellular altered foci (HAF) that expressed the placental form of glutathione S-transferase. AAF induced HAF in the high exposure group, 9-fold at 8 weeks and 170-fold at 12 weeks compared to controls. In rats maintained on PB for 24 weeks after exposure, the multiplicity of HAF increased in controls and comparably in the low and mid exposure groups, but remained at the about the same high level in the high exposure group. The high exposure produced a substantial incidence of benign neoplasms by 12 weeks, and with promotion by 36 weeks, all rats developed hepatocellular neoplasia. In the mid exposure group, only one adenoma occurred at 36 weeks in 17 rats, while in the low exposure group, no liver tumor occurred in 23 rats. Thus, these findings document nonlinearities for some of the effects of AAF, with supralinear effects at the high exposure for cell proliferation and induction of HAF, and a no-observed-effect level for induction of promotable liver neoplasms at the lowest cumulative exposure of 0.126 mmol/kg, in spite of the formation of DNA adducts. We conclude that the effects of this DNA-reactive hepatocarcinogen leading to initiation exhibit nonlinearities and possible thresholds.     

Enhancing effect of O6-alkylguanine derivatives on chloroethylnitrosourea cytotoxicity toward tumor cells

O6-Alkylguanine derivatives are well known as chemical modulators of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT). Depletion of the enzyme by these derivatives leads to increase sensitivity of tumor cells to chloroethylnitrosoureas. We tested the effect of O6-methylguanine, O6-benzylguanine, O6-(p-methylbenzyl)guanine, O6-(p-chlorobenzyl)guanine, O6-(p-methoxybenzyl)guanine, O6-methylhypoxanthine and O6-benzylhypoxanthine on the sensitivity of tumor cell lines to 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3- nitrosourea hydrochloride (ACNU) using a colorimetric cytotoxicity assay. The sensitivity of MGMT-proficient tumor cells including HeLA S3, C6-1, C6-2/ACNU, U-138 MG and U-373 MG cells was greatly enhanced by 2 hr pretreatment of 10-100 microM O6-benzylguanine, O6-(p-methylbenzyl)guanine and O6-(p-chlorobenzyl)guanine, but not by O6-methylguanine or O6-methylhypoxanthine. O6-(p-methylbenzyl)guanine moderately sensitized the 2 cell lines, HeLa S3 and C6-1, tested in our study to ACNU cytotoxicity. O6-Benzylhypoxanthine at the high concentration (100 microM) sensitized, to some extent, 3 MGMT-proficient cell lines. Lesser degrees of enhancement by the O6-benzylguanine derivatives were noted in MGMT-deficient tumor cells. Biological effects of O6-alkylguanine derivatives on enhancing ACNU cytotoxicity of tumor cells suggest that the exocyclic 2-amino and O6-benzyl groups in O6-benzylguanine skeleton are both essential for the inhibition of MGMT activity.     

Inhibitory effects of lemon grass (Cymbopogon citratus Stapf) on formation of azoxymethane-induced DNA adducts and aberrant crypt foci in the rat colon

The 80%-ethanol extract of lemon grass (Cymbopogon citratus Stapf), a medicinal plant in Thailand, has been reported to be antimutagenic against various known mutagens in the Salmonella mutation assay. To investigate chemoprevention in an animal carcinogenesis model, we examined inhibitory effects of the lemon grass extract on the formation of azoxymethane (AOM)-induced DNA adducts and aberrant crypt foci (ACF) in the rat colon. One week after the start of the treatment with lemon grass extract at doses of 0.5 or 5 g/kg body wt by gavage, F344 rats received two s.c. injections of 15 mg of AOM per kg body weight at 1 week apart. For DNA adduct analysis of the colon and liver, the rats were killed 12 h after the second AOM injection. The DNA from the liver and colon were used for O6-methylguanine and N7-methylguanine analysis. For ACF analysis in the initiation stage, AOM-injected rats were continuously treated with lemon grass extract and were killed 3 weeks after the second AOM injection. For analysis in the promotion stage the treatment with the lemon grass extract (0.5 g/kg) started 2 weeks after the second AOM injection and continued for 12 weeks until the animals were killed. Lemon grass treatment significantly inhibited DNA adduct formation in both the colonic mucosa and the muscular layer but not in the liver. In addition, lemon grass extract treatment significantly inhibited ACF formation in both the initiation stage and the promotion stage. Especially in the promotion stage, lemon grass treatment inhibited the formation of larger ACF (with four or more crypts per focus), which was predictive of tumor incidence. Furthermore, lemon grass extract inhibited fecal beta-glucuronidase competitively and had antioxidant activity. These results suggest that the lemon grass extract inhibits the release of activated aglycon, methylazoxymethanol, from a glucuronide conjugate in the colon, and decreases the DNA adducts and ACF formation in the rat colon.     

Protective effects of green tea on hepatotoxicity, oxidative DNA damage and cell proliferation in the rat liver induced by repeated oral administration of 2-nitropropane

To evaluate the benefit of green tea in mitigating hazards caused by repeated exposure of 2-nitropropane (2NP), we examined the effects of the tea on toxic indices, oxidative DNA damage and cell proliferation in the liver of 2NP-treated rats. Male Fischer 344 rats were administered, by gastric intubation, a total of six doses of 60 mg/kg 2NP(L), or alternatively two doses of 90 mg/kg and then four doses of 120 mg/kg 2NP(H) during 2 weeks. Green tea infusion was given to the rats as drinking water 1 week before the 2NP treatments and throughout the experiment. Significant elevation of hepatotoxic indices was evident in the 2NP(H)-treated group, such as an increase of serum glutamic-oxaloacetic transaminase (GOT) activity and of hepatic lipid peroxidation, together with a decrease in hepatic glycogen and serum triglyceride, and degenerative changes in the hepatocytes. A dose-related increase was observed in oxidative DNA damage and cell proliferation in the liver. Green tea effectively inhibited all of above changes induced by 2NP treatment, suggesting that tea intake may be effective for preventing the hepatic injuries after chronic exposure to 2NP.     

Comparative study of the formation and repair of O6-methylguanine in humans and rodents treated with dacarbazine

The mutagenic, carcinogenic and cytotoxic activity of dacarbazine, a drug employed in cancer chemotherapy, may be related to the induction in DNA of O6-methylguanine (O6-meG), a quantitatively minor but biologically important lesion. In the present study the kinetics of O6-meG formation and repair in blood leukocyte DNA were examined in 20 Hodgkins lymphoma patients treated i.v. with 180 +/- 13 (mean +/- SD) mg/m2 dacarbazine and compared with those observed in various tissues of rodents treated with different doses of the drug. In Hodgkin's lymphoma patients adduct levels reached a value of 0.27 +/- 0.14 fmol/microgram DNA 2 h after dacarbazine administration, while the rate of subsequent loss suggested an adduct half-life of < or = 30 h. Measurement of adduct levels in the same individuals after successive courses of treatment spaced 3 weeks apart (up to 10 treatment courses) demonstrated a consistent individual response and statistical analysis of variance confirmed that intra-individual variation in adduct accumulation after a given dose of dacarbazine accounted for only 5% of the total variance observed. In contrast, inter-individual variation accounted for 70% of the observed variance, with adduct levels 2 h after drug treatment varying approximately 7.5-fold among adduct-positive individuals. No significant depletion of lymphocyte O6-alkylguanine-DNA alkyltransferase (AGT) occurred after patient treatment with dacarbazine. No significant relationship between adduct levels and clinical response to treatment was observed. In rats treated with single or multiple doses of dacarbazine causing varying degrees of AGT depletion the highest levels of O6-meG were seen in the liver, followed by the lymph nodes, bone marrow and blood leukocytes, which showed up to approximately 2-fold lower levels. A similar tissue distribution was also observed in mice and in a single rabbit. These observations suggest that O6-meG levels assayed in blood leukocytes of therapeutically treated humans reflect those present in the -lymph nodes (target tissue for chemotherapy) and the bone marrow (target tissue for leukaemogenesis) and may be utilized as a measure of the drug dose reaching these tissues. The quantitative data reported in this study show that under conditions of no depletion of AGT O6-meG accumulates in blood leukocyte DNA of humans at a rate similar to that observed in rats, suggesting that human susceptibility to any O6-meG-mediated genotoxic effects of dacarbazine may be comparable with that of the rat.     

Xenomorphic hepatocellular precursors and neoplastic progression of tigroid cell foci induced in rats with low doses of N-nitrosomorpholine

Tigroid cell foci (TCF) are a well-defined entity induced in rat liver by chemical carcinogens, their significance for hepatocarcinogenesis being controversial. Using cytomorphological, cytochemical and morphometric approaches, we studied the evolution and fate of TCF sequentially from 7 to 110 weeks in groups of 50 male Sprague-Dawley rats, which remained untreated or received N-nitrosomorpholine (NNM) orally at concentrations of 3 and 1 mg/kg body wt/day for 7 and up to 75 weeks, respectively. An increased incidence of hepatocellular neoplasms developed in exposed animals compared with controls, which was significant for adenomas at both dose levels, and for carcinomas (HCC) after the longer exposure to the lower dose level (P < 0.0001). TCF appeared frequently in addition to other types of proliferative foci of altered hepatocytes (FAH) including clear/acidophilic and mixed cell foci (MCF) in NNM-treated and rarely in untreated rats. Striking similarities in the cellular phenotypes of TCF and many hepatocellular neoplasms indicated the potential of TCF for progression to both adenomas and carcinomas. TCF emerged from xenomorphic cell foci (XCF), which consisted of hypertrophied hepatocytes typically presenting an enlarged nucleus, abundant glycogen, smooth and rough endoplasmic reticulum, altered activities of several enzymes of carbohydrate metabolism and an increased cell proliferation (P < 0.001) compared with the extrafocal parenchyma. TCF shared many features with XCF, but their basophilia and proliferative activity was higher. The number of FAH appearing at the two dose levels of NNM was similar but the average size of TCF and MCF was frequently higher at late time points in the group developing a significantly higher incidence of HCC, which suggests a pronounced acceleration of neoplastic conversion in established preneoplastic cell populations rather than the induction of additional FAH by sustained effects of low doses of carcinogens.     

Recent bacterial and viral infection is a risk factor for cerebrovascular ischemia: clinical and biochemical studies

Inbred mice vary in susceptibility to colon carcinogens such as 1,2-dimethylhydrazine (DMH). Differential susceptibility may depend, in part, on formation of promutagenic DNA methyl adducts within target colonic mucosa. The present study was undertaken to evaluate the extent of DNA adduct formation in susceptible (SWR) and resistant (AKR) mice acutely exposed to the colon carcinogen azoxymethane (AOM), a direct metabolite of DMH. In the first experiment, 8-week-old SWR and AKR mice were treated i.p. with 20 mg/kg AOM and sacrificed 6 h later. DNA was isolated from distal colon and liver, and O6-methylguanine (O6-MeGua) adduct levels were assessed by immunoslot blot (ISB) analysis, using a monospecific antibody raised against O6-methyldeoxyguanosine. HPLC-fluorescence detection was also used to quantitate 06-MeGua and 7-methylguanine (7-MeGua), and to generate standard curves. At 6 h, both O6-MeGua and 7-MeGua were significantly higher (2- to 3-fold, p < 0.05) in AKR colon, while an opposite pattern was found in liver. In Experiment 2, mice were injected with AOM (20 mg/kg) and euthanized 12 and 48 h later. At 12 h, O6-MeGua levels were higher in colons (1.4-fold) of SWR mice. Forty-eight hours after treatment, however, adduct levels in colon were markedly (5-fold) reduced in SWR but were unchanged from 12 h in AKR. To further compare activation of AOM in both strains, colon microsomes were incubated with AOM and calf thymus DNA. Comparable levels of O6-MeGua were detected by ISB, demonstrating equivalent metabolic capacity in both SWR and AKR mice. These studies suggest that differential susceptibility to AOM-induced colon carcinogenesis is not based on initial target tissue DNA alkylation and unlikely to depend on differential metabolic capacity.     

Free radical-induced megamitochondria formation and apoptosis

Pathophysiological meaning and the mechanism of the formation of megamitochondria (MG) induced under physiological and pathological conditions remain obscure. We now provide evidence suggesting that the MG formation may be a prerequisite for free radical-mediated apoptosis. MG were detected in primary cultured rat hepatocytes, rat liver cell lines RL-34 and IAR-20 and kidney cell line Cos-1 treated for 22 h with various chemicals known to generate free radicals: hydrazine, chloramphenicol, methyl-glyoxal-bis-guanylhydrazone, indomethacin, H2O2, and erythromycin using a fluorescent dye Mito Tracker Red CMXRos (CMXRos) for confocal laser microscopy and also by electron microscopy. Remarkable elevations of the intracellular level of reactive oxygen species (ROS), monitored by staining of cells with a fluorescent dye carboxy-H2-DCFDA, were detected before MG were formed. Prolongation of the incubation time with various chemicals, specified above, for 36 h or longer has induced distinct structural changes of the cell, which characterize apoptosis: condensation of nuclei, the formation of apoptotic bodies, and the ladder formation. Cells treated with the chemicals for 22 h were arrested in G1 phase, and apoptotic sub-G1 populations then became gradually increased. The membrane potential of MG induced by chloramphenicol detected by CMXRos for flow cytometry was found to be decreased compared to that of mitochondria in control cells. Rates of the generation of H2O2 and O2- from MG isolated from the liver of rats treated with chloramphenicol or hydrazine were found to be lower than those of mitochondria of the liver of control animals. We suggest, based on the present results together with our previous findings, that the formation of MG may be an adaptive process at a subcellular level to unfavorable environments: when cells are exposed to excess amounts of free radicals mitochondria become enlarged decreasing the rate of oxygen consumption. Decreases in the oxygen consumption of MG may result in decreases in the rate of ROS production as shown in the present study. This will at the same time result in decreases in ATP production from MG. If cells are exposed to a large amount of free radicals beyond a certain period of time, lowered intracellular levels of ATP may result in apoptotic changes of the cell.     

Formation and accumulation of DNA ethenobases in adult Sprague-Dawley rats exposed to vinyl chloride

DNA ethenobases are promutagenic lesions formed by carcinogens such as vinyl chloride (VC). Their formation was investigated in 6-week old, male Sprague-Dawley rats exposed to 500 p.p.m. VC by inhalation (4 h/day, 5 days/ week) for 1, 2, 4 or 8 weeks and in 7- and 14-week old, matched control animals. 1,N6-Ethenoadenine (epsilon A) and 3, N4-ethenocytosine (epsilon C) deoxyribonucleotides were analysed by immunoaffinity purification and 32P-postlabelling. This postlabelling method was compared with a radio-immunoassay method, which yielded similar results. Background levels of ethenobases were found in DNA from the liver, lungs, kidneys and circulating lymphocytes of unexposed, control rats. In the liver, the following background molar ratios of ethenobase to parent base in DNA were detected (mean values x 10(-8)): epsilon A/A, 0.04-0.05; epsilon C/C, 0.06-0.07. In the lungs, kidneys and circulating lymphocytes, background levels of epsilon A and epsilon C ranged from 1.7 to 4.2 x 10(-8) and from 4.8 to 11.2 x 10(-8), respectively. Following a 5-day exposure to VC, a significant increase of epsilon A and epsilon C was measured in hepatic DNA from rats sacrificed immediately after treatment. Further, a dose-dependent increase of both etheno adducts was observed in liver DNA of VC-treated rats. Compared to the 5-day exposure, approximately 4-fold higher levels of epsilon A and epsilon C were observed in the liver of animals after 8 weeks of exposure. In contrast, there was an accumulation of epsilon C but not of epsilon A in lungs and kidneys. In circulating lymphocytes, no significant increase of ethenobase levels above control values was observed after 2 months of exposure to VC. Both etheno adducts were found to be persistent in liver DNA, after 2 months following the termination of VC exposure. These results further support the notion that DNA etheno-bases are critical lesions in VC-induced carcinogenesis. The possible contribution of lipid peroxidation products that also yield ethenobases, on the formation and persistence of these DNA adducts, remains to be clarified.     

Granulocyte colony-stimulating factor administration reverses cadmium-associated inhibition of hepatocyte regeneration

OBJECTIVE: To document whether the administration of granulocyte colony-stimulating factor (G-CSF) enhances the impaired regenerative response of hepatocytes to partial hepatectomy (PH), in cadmium-pretreated partially hepatectomized rats. MATERIALS AND METHODS: Rats were injected intraperioneally with 2.5 mg CdCl2/kg body weight, 24h before PH. G-CSF (1500 or 150 micrograms/kg body weight) or saline was administered intraperitoneally in cadmium-pretreated partially hepatectomized rats at the same time as PH. The liver regenerative process was estimated 24h after PH. [3H] thymidine incorporation into liver DNA, liver thymidine kinase (TK) activity, mitotic index and proliferating cell nuclear antigen (PCNA) immunostaining were used as indices of hepatocyte proliferation. RESULTS: G-CSF administration in cadmium-pretreated partially hepatectomized rats restored the suppressed DNA biosynthesis and TK activity (P < 0.001), to levels similar to those found in rats that were partially hepatectomized only. The mitotic index and the percentage of PCNA positive nuclei in hepatocytes were also enhanced in G-CSF administered cadmium-pretreated partially hepatectomized groups of rats. CONCLUSION: The administration of G-CSF triggers events that restore the impaired liver regeneration in this model of reduced hepatocyte proliferation.     

A non-bile duct origin for intestinal crypt-like ducts with periductular fibrosis induced in livers of F344 rats by chloroform inhalation

To evaluate the toxic effects of prolonged exposure to chloroform vapors, female and male F344 rats were exposed to 0, 2, 10, 30, 90 and 300 p.p.m. chloroform by inhalation for 7 or 5 days/week for up to 13 weeks. The purpose of this study was to characterize a lesion that occurred in the livers of rats in the 300 p.p.m. exposure groups. Atypical glandular structures lined by intestinal-like epithelium and surrounded by dense connective tissue occurred in the livers of rats exposed to strongly hepatotoxic atmospheric concentrations of chloroform. Bile duct bromodeoxyuridine labeling indices as well as observations of the locations of the early lesions at the 3 and 6 week time points indicate that these lesions arose from a population of cells remote from the bile ducts. We refer to these lesions as intestinal crypt-like ducts with periductular fibrosis to distinguish them from true cholangiofibrosis. Here, intestinal crypt-like ducts with periductular fibrosis were seen only in rats exposed to 300 p.p.m. chloroform, and the multiplicity and severity of the lesions were greater in the right liver lobe. The lesion only occurred in association with liver necrosis and dramatic increases in hepatocyte labeling indices, while labeling indices in bile ducts in the same animals were not significantly different from controls. There was a treatment-related increase of transforming growth factor-alpha immunoreactivity in hepatocytes, bile duct epithelium, bile canaliculi and oval cells, and an increase in transforming growth factor-beta immunoreactivity in hepatocytes, bile duct epithelium and intestinal crypt-like ducts. Thus, intestinal crypt-like ducts with periductular fibrosis appeared to develop from a population of cells unrelated to bile ducts. Also, they occurred only in animals exposed to chloroform concentrations that induced significant hepatocyte necrosis and regenerative cell proliferation and were associated with increased growth factor expression or uptake.     

Strand- and sequence-specific attenuation of N-methyl-N'-nitro-N-nitrosoguanidine-induced G.C to A.T transitions by expression of human 6-methylguanine-DNA methyltransferase in Chinese hamster ovary cells

The effect of human O6-methylguanine-DNA methyltransferase (MGMT) on the cytotoxicity, the mutagenicity, and the specific kinds of base substitutions induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were examined in non-MGMT transfected Chinese hamster ovary cells (CHOM cells) and in those cells which had been transfected with human MGMT complementary DNA (AGT cells). AGT cells containing a high level of human MGMT activity were markedly more resistant to the cytotoxic and mutagenic effects of MNNG than CHOM cells which had no detectable MGMT activity. The dosages of MNNG which reduced to 50% of colony forming ability were estimated to be 0.8 microM for CHOM and 10 microM for AGT cells. The induction frequency of 6-thioguanine-resistant cells was significantly declined in AGT cells. At 4 microM MNNG, this frequency was declined from 273 mutants/10(6) viable CHOM cells to 13 mutants/10(6) viable AGT cells. The entire coding region of the hypoxanthine (guanine) phosphoribosyltransferase (hprt) gene in 37 AGT and 22 CHOM mutants was characterized by direct sequencing of the mRNA-polymerase chain reaction-amplified complementary DNA. Base changes at the intron-exon boundaries of the hprt DNA in the splicing mutants were further examined. Those results indicated that G to A transitions were significantly reduced in MNNG-treated AGT cells (chi 2 test, P < 0.001), suggesting that O6-methylguanine was repaired error free by human MGMT. In contrast, no difference arose in the frequencies of T to C transitions induced by MNNG in these two populations. All of the G to A transitions induced in AGT cells were located on the nontranscribed strand, assuming that the causative lesion was O6-methylguanine (P < 0.05). Such a strand specificity was not observed in CHOM mutants. Most of the G to A transitions observed in CHOM mutants were located at the middle guanine of 5'-GGPu sequences. Transitions observed at these sites, particularly 5'-GGG, were significantly reduced in AGT mutants (P < 0.05). Our results have suggested that human MGMT specifically repairs O6-methylguanine with a preference to remove those located on the transcribed strand and middle guanine of 5'-GGG.