Ornithine decarboxylase inhibition downregulates multiple pathways involved in the formation of precancerous lesions of esophageal squamous cell cancer.

The high incidence and mortality of esophageal squamous cell cancer (ESCC) is a major health problem worldwide. Precancerous lesions of ESCC may either progress to cancer or revert to normal epithelium with appropriate interventions; the bidirectional instability of the precancerous lesions of ESCC provides opportunities for intervention. Reports suggest that the upregulation of ornithine decarboxylase (ODC) is closely related to carcinogenesis. In this study, we investigated whether ODC may act as a target for chemoprevention in ESCC. Immunohistochemistry (IHC) assays indicate that ODC expression is higher in esophageal precancerous lesions compared with normal tissue controls. Its overexpression promotes cell proliferation and transformation of normal esophageal epithelial cells, and its activity is increased after N-nitrosomethylbenzylamine (NMBA) induction in Shantou human embryonic esophageal cell line (SHEE) and human immortalized cells (Het1A) cells. In addition, p38 α, extracellular regulated kinase (ERK1/2) in the mitogen-activated protein kinase pathway and protein kinase B (AKT)/mammalian target of rapamycin (mTOR)/ribosomal protein S6 kinase (p70S6K) pathways are activated in response to NMBA treatment. Difluoromethylornithine (DFMO) is an ODC inhibitor, which inhibits NMBA-induced activation of p38 α, ERK1/2 and AKT/mTOR/p70S6K pathways; this has been verified by Western blotting. DFMO was also found to suppress the development of esophageal precancerous lesions in an NMBA-induced rat model; IHC demonstrated p38 α, ERK1/2, and AKT/mTOR/p70S6K pathways to be downregulated in these rats. These findings indicate the mechanisms by which ODC inhibition suppresses the development of esophageal precancerous lesions by downregulating p38 α, ERK1/2, and AKT/mTOR/p70S6k signaling pathways, ODC may be a potential target for chemoprevention in ESCC.

Mismatch repair systems might facilitate the chromosomal recombination induced by N-nitrosodimethylamine, but not by N-nitrosodiethylamine, in Drosophila.

Mismatch repair (MMR) systems play important roles in maintaining the high fidelity of genomic DNA. It is well documented that a lack of MMR increases the mutation rate, including base exchanges and small insertion/deletion loops; however, it is unknown whether MMR deficiency affects the frequency of chromosomal recombination in somatic cells. To investigate the effects of MMR on chromosomal recombination, we used the Drosophila wing-spot test, which efficiently detects chromosomal recombination. We prepared MMR (MutS)-deficient flies (spel1(-/-)) using a fly line generated in this study. The spontaneous mutation rate as measured by the wing-spot test was slightly higher in MutS-deficient flies than in wild-type (spel1(+/-)) flies. Previously, we showed that N-nitrosodimethylamine (NDMA)-induced chromosomal recombination more frequently than N-nitrosodiethylamine (NDEA) in Drosophila. When the wing-spot test was performed using MMR-deficient flies, unexpectedly, the rate of NDMA-induced mutation was significantly lower in spel1(-/-) flies than in spel1(+/-) flies. In contrast, the rate of mutation induced by NDEA was higher in spel1(-/-) flies than in spel1(+/-) flies. These results suggest that in Drosophila, the MutS homologue protein recognises methylated DNA lesions more efficiently than ethylated ones, and that MMR might facilitate mutational chromosomal recombination due to DNA double-strand breaks via the futile cycle induced by MutS recognition of methylated lesions.

The effect of ethanol and N-nitrosodimethylamine on the iNOS-dependent NO production in human neutrophils. Role of NF-κB.

1. The objective of study was to determine the influence of ethanol and/or N-nitrosodimethyloamine (NDMA) on the inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production by human neutrophils and determination of the role of NF-κB in this process. 2. Isolated polymorphonuclear leukocytes (PMNs) derived from 15 human volunteers were incubated in the presence of ethanol and/or NDMA. Expression of the tested proteins were evaluated using the Western blot method. Total NO metabolites was assayed in the cell cultures by Griess reaction. 3. In neutrophils exposed to ethanol or NDMA was observed an increased NF-κB-dependent NO production mediated by iNOS with the contribution of MAP kinases: p38 and JNK. An inhibiting effect of NF-κB signaling pathway on the MAP kinases was observed, which are involved in the iNOS-dependent NO production. By the simultaneous effect, ethanol and NDMA caused stronger generation of NO by neutrophils without the contribution of iNOS. Inhibition of NF-κB in cells simultaneously exposed to the xenobiotics caused a decreased expression of MAP kinases. 4. Individual and simultaneous effect of ethanol and NDMA may cause disorders in the response of immune system. However, the joint effect of the tested substances results in uncontrolled interactions, leading to cascading disorders of signal transduction.

Bovine milk-derived α-lactalbumin prevents hepatic fibrosis induced by dimethylnitrosamine via nitric oxide pathway in rats.

The present study was designed to evaluate the hepatoprotective potential of α-lactalbumin (αLA) against dimethylnitrosamine (DMN)-induced toxic insults in the rat liver. The liver damage was induced in rats by the repeated administration of DMN (10 mg/kg, i.p.) on three consecutive days per week for three weeks. The rats were maintained on either a standard AIN-93 M or αLA-enriched diet starting one week before the DMN injection until the termination of the experiment. The DMN treatment produced a progressive increase in the plasma markers (aspartate aminotransferase, alanine aminotransferase, total bililbin, hyarulonic acid, and matrix metalloproteinase-2) in 28 days after the first DMN injection. Dietary treatment with αLA significantly reduced the DMN-induced damage toward normalcy. NＧ-nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor, significantly attenuated the hepatoprotective effect of αLA. These findings show that αLA has a marked suppressive effect on hepetic fibrosis through a nitric oxide-mediated mechanism.

Dicliptera Chinensis polysaccharides target TGF-ß/Smad pathway and inhibit stellate cells activation in rats with dimethylnitrosamine-induced hepatic fibrosis.

This study aims to study impact of Dicliptera chinensis polysaccharide (DCP) on hepatic fibrosis (HF) and activation of hepatic stellate cells (HSCs). Liver fibrosis model was induced by intraperitoneal injection of dimethyl nitrosamines (DMN) in rat. Rats in treatment group were administrated with different concentrations of DCP (0, 100, 300 mg/kg) by intraperitoneal injection. Hematoxylin and eosin (H&E) and Masson's trichrome staining were used to assess histo-pathological change. α-SMA, TGF-ß1 and pSmad 2/3 were assayed by immuno-histochemistry. HSC-T6 cells were stimulated by recombined rat TGF-ß1 (1 ng/mL) to simulate an activating model in vitro and then interfered with DCP (concentration of 0, 25, 50, 100, 200, 400 µg/ml). MTT assay was used to determine cell proliferation and western blotting was used to detect α-SMA and pSmad 2/3 expression. Results demonstrated that DCP alleviated DMN-induced liver fibrosis in rat and significantly down-regulated TGF-ß1 expression, pSmad2/3 and α-SMA in liver tissue in a dose-dependent way. DCP inhibited proliferation and activation of TGF-ß1-stimulated HSC-T6 in vitro and significantly down-regulated α-SMA and pSmad2/3 expression. In conclusion, this study revealed that DCP attenuates progression of liver fibrosis through suppressing TGF-ß/Smad pathway. DCP is a potential botanical polysaccharide to management liver fibrosis.

Role of ERK1/2 kinase in the expression of iNOS by NDMA in human neutrophils.

Potential role of ERK1/2 kinase in conjunction with p38 in the regulation of inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production, and superoxide anion generation by human neutrophils (PMNs) exposed to N-nitrosodimethylamine (NDMA) was determined. Increased synthesis of NO due to the involvement of iNOS in neutrophils exposed to NDMA was observed. In addition, intensified activation of ERK1/2 and p38 kinases was determined in these cells. Inhibition of kinase regulated by extracellular signals (ERK1/2) pathway, in contrast to p38 pathway, led to an increased production of NO and expression of iNOS in PMNs. Moreover, as a result of inhibition of ERK1/2 pathway, a decreased activation of p38 kinase was observed in neutrophils, while inhibition of p38 kinase did not affect activation of ERK1/2 pathway in these cells. An increased ability to release superoxide anion by the studied PMNs was observed, which decreased after ERK1/2 pathway inhibition. In conclusion, in human neutrophils, ERK1/2 kinase is not directly involved in the regulation of iNOS and NO production induced by NDMA; however, the kinase participates in superoxide anion production in these cells.

Zinc Oxide Nanoparticles Ameliorate Dimethylnitrosamine-Induced Renal Toxicity in Rat.

Dimethylnitrosamine (DMN) is an established carcinogen. It is toxic to several organs, viz., the liver, kidney, and lungs, and immune system. Several drugs have been used in the past to modulate its toxicity using experimental animal models. The present study was designed to investigate the effect of zinc oxide nanoparticles (ZnONPs) on renal toxicity caused by DMN in laboratory rat. Since oxidative mechanisms are mainly involved in its toxicity, the proposed study focuses on the amelioration of oxidative stress response by ZnONPs, if any. The present results show that administration of ZnONPs (50 mg/kg body weight/rat) to DMN (2 µl/100 g body weight/rat)-treated rats diminuted the concentration of malonaldehyde, H2O2, and NO in the kidney. However, reduced glutathione (GSH) concentration increased after ZnONP treatment. Results on glutathione S-transferase and glutathione peroxidase favored its antioxidative effects. These results are supported by the recovery of oxidative DNA damage and less pronounced histopathological changes in the kidney. It is hypothesized that ZnONPs might be toxic to renal tissue; however, its strong therapeutic/antioxidative potential helps in ameliorating DMN-induced renal toxicity in rat.

Design and optimization of cranberry extract loaded bile salt augmented liposomes for targeting of MCP-1/STAT3/VEGF signaling pathway in DMN-intoxicated liver in rats.

Cranberry extract (CBE) is a major source of the antioxidant polyphenolics but suffers from limited bioavailability. The goal of this research was to encapsulate the nutraceutical (CBE), into bile salt augmented liposomes (BSALs) as a promising oral delivery system to potentiate its hepatoprotective impact against dimethylnitrosamine (DMN) induced liver injury in rats. The inclusion of bile salt in the liposomal structure can enhance their stability within the gastrointestinal tract and promote CBE permeability. CBE loaded BSALs formulations were fabricated utilizing a (23) factorial design to explore the impact of phospholipid type (X1), phospholipid amount (X2), and sodium glycocholate (SGC) amount (X3) on BSALs properties, namely; entrapment efficiency percent, (EE%); vesicle size, (VS); polydispersity index; (PDI); zeta potential, (ZP); and release efficiency percent, (RE%). The optimum formulation (F1) exhibited spherical vesicles with EE% of 71.27 ± 0.32%, VS; 148.60 ± 6.46 nm, PDI; 0.38 ± 0.02, ZP; -18.27 ± 0.67 mV and RE%; 61.96 ± 1.07%. Compared to CBE solution, F1 had attenuated DMN-induced hepatic injury, as evidenced by the significant decrease in serum level of ALT, AST, ALP, MDA, and elevation of GSH level, as well as SOD and GPX activities. Furthermore, F1 exhibited an anti-inflammatory character by suppressing TNF-α, MCP-1, and IL-6, as well as downregulation of VEGF-C, STAT-3, and IFN-γ mRNA levels. This study verified that when CBE was integrated into BSALs, F1, its hepatoprotective effect was significantly potentiated to protect the liver against DMN-induced damage. Therefore, F1 could be deliberated as an antioxidant, antiproliferative, and antifibrotic therapy to slow down the progression of hepatic damage.

Aromatic C-nitrosation of a bioactive molecule. Nitrosation of minoxidil.

Minoxidil (2,4-diamino-6-(piperidin-1'-yl)pyrimidine N(3)-oxide; CASRN 38304-91-5) is a bioactive molecule with several nitrosatable groups widely used as an antihypertensive and antialopecia agent. Here the nitrosation of minoxidil was investigated. The conclusions drawn are as follows: (i) In the pH = 2.3-5.0 range, the minoxidil molecule undergoes aromatic C-nitrosation by nitrite. The dominant reaction was C-5 nitrosation through a mechanism that appears to consist of an electrophilic attack on the nitrosatable substrate by H(2)NO(2)(+)/NO(+), followed by a slow proton transfer; (ii) the reactivity of minoxidil as a C-nitrosatable substrate proved to be 7-fold greater than that of phenol, this being attributed to the preferred para- and ortho-orientations of the two -NH(2) groups at positions 2 and 4 of the minoxidil molecule, which activate electrophilic substitution in the C-5 position through their mesomeric effect. The N-nitrosominoxidil resulting from the nitrosation could be potentially harmful to the minoxidil users.

Mucin-producing hamster cholangiocarcinoma cell line, Ham-2, possesses the aggressive cancer phenotypes with liver and lung metastases.

Cholangiocarcinoma (CCA) is an aggressive bile duct cancer. Opisthorchis viverrini (O. viverrini) infection is a significant cause of CCA in the Greater Mekong subregion. Currently, there is no standard chemotherapeutic regimen for CCA. A unique hamster carcinogenesis model of O. viverrini-associated CCA was established. Molecular targets identified from the hamster CCA-comparative model are valuable for target identification and validation. Hamster CCA was induced by the administration of O. viverrini metacercariae and N-nitrosodimethylamine. Hamster-derived cancer cells were isolated and continuously cultured for more than 6 months. Ham-2 cell line was established and characterized in vitro and in vivo. Ham-2 exhibited chromosome hyperploidy. A comparative study with previously established cell line, Ham-1, demonstrated that Ham-2 acquired slower growth, higher adhesion, higher migration, and resistance to doxorubicin and 5-fluorouracil (5-FU). In BALB/c Rag-2/Jak3 double-deficient (BRJ) mice, Ham-2 subcutaneous transplantation formed mucin-producing cancers, which morphologically resemble human tubular cholangiocarcinoma. Intravenous-injected Ham-2 established the metastatic nodules in the lungs and livers of BRJ mice. Altogether, a new hamster cholangiocarcinoma cell line, Ham-2, which acquired more aggressive phenotypes in vitro and in vivo, was established. This cell line might be a valuable tool for comparative drug target identification and validation.

Kupffer cells are associated with apoptosis, inflammation and fibrotic effects in hepatic fibrosis in rats.

Hepatocellular apoptosis, hepatic inflammation, and fibrosis are prominent features in chronic liver diseases. However, the linkage among these processes remains mechanistically unclear. In this study, we examined the apoptosis and activation of Kupffer cells (KCs) as well as their pathophysiological involvement in liver fibrosis process. Hepatic fibrosis was induced in rats by dimethylnitrosamine (DMN) or carbon tetrachloride (CCl4) treatment. KCs were isolated from normal rats and incubated with lipopolysaccharide (LPS) or from fibrotic rats. The KCs were stained immunohistochemically with anti-CD68 antibody, a biomarker for KC. The level of expression of CD68 was analyzed by western blot and real-time PCR methods. The apoptosis and pathophysiological involvement of KCs in the formation of liver fibrosis were studied using confocal microscopy. The mRNA and protein expression of CD68 were significantly increased in DMN- and CCL4-treated rats. Confocal microscopy analysis showed that CD68-positive KCs, but not α-smooth muscle actin (SMA)-positive cells, underwent apoptosis in the liver of DMN- and CCL4-treated rats. It was also revealed that the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and CD68-double-positive apoptotic KCs located in the portal or fibrotic septa area were situated next to hepatic stellate cells (HSCs). Tumor necrosis factor-α (TNF-α) and KC co-localized in the liver in the neighbor of HSCs. The double α-SMA- and collagen type I-positive cells predominantly existed in fibrotic septa, and those cells were co-localized clearly with CD68-positive cells. Interestingly, some CD68 and Col (1) double positive, but completely negative for α-SMA, were found in the portal areas and hepatic sinusoids; this phenomenon was also validated in primary isolated KCs after 6 h LPS exposure or fibrotic rats in vitro. These results show that KCs are associated with hepatocellular apoptosis, inflammation, and fibrosis process in a liver fibrosis models.

Induction of expression of iNOS by N-nitrosodimethylamine (NDMA) in human leukocytes.

The aim of this study was to assess the influence of N-nitrosodimethylamine (NDMA) on expression of inducible nitric oxide synthase (iNOS), as well as production of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) by human neutrophils (PMN) and peripheral blood mononuclear cells (PBMC), and the participation of the p38 MAPK kinase in this process. Furthermore, the ability of neutrophils to release superoxide anion was determined. The influence of N-nitrosodimethylamine on iNOS expression was determined in isolated PMN and PBMC cells from peripheral blood of healthy individuals. The mononuclear cells showed higher sensitivity to NDMA. Moreover, cytotoxic effect of NDMA can be influenced in some way by the impact of this xenobiotic on nitric oxide and superoxide anion release from human leukocytes. Furthermore, increased generation of these radicals by human leukocytes suggest that neutrophils and mononuclear cells that are exposed to NDMA activity can play a key role in endogenous NDMA generation. However the relationship between iNOS expression and phospho-p38 MAPK in neutrophils and mononuclear cells shows that p38 MAPK pathway participates in induction of iNOS expression in the presence of NDMA.

Molecular mechanisms in the pathogenesis of N-nitrosodimethylamine induced hepatic fibrosis.

Hepatic fibrosis is marked by excessive synthesis and deposition of connective tissue proteins, especially interstitial collagens in the extracellular matrix of the liver. It is a result of an abnormal wound healing in response to chronic liver injury from various causes such as ethanol, viruses, toxins, drugs, or cholestasis. The chronic stimuli involved in the initiation of fibrosis leads to oxidative stress and generation of reactive oxygen species that serve as mediators of molecular events involved in the pathogenesis of hepatic fibrosis. These processes lead to cellular injury and initiate inflammatory responses releasing a variety of cytokines and growth factors that trigger activation and transformation of resting hepatic stellate cells into myofibroblast like cells, which in turn start excessive synthesis of connective tissue proteins, especially collagens. Uncontrolled and extensive fibrosis results in distortion of lobular architecture of the liver leading to nodular formation and cirrhosis. The perpetual injury and regeneration process could also results in genomic aberrations and mutations that lead to the development of hepatocellular carcinoma. This review covers most aspects of the molecular mechanisms involved in the pathogenesis of hepatic fibrosis with special emphasize on N-Nitrosodimethylamine (NDMA; Dimethylnitorsmaine, DMN) as the inducing agent.

Sequential assessment of the intrahepatic expression of epidermal growth factor and transforming growth factor-beta1 in hepatofibrogenesis of a rat cirrhosis model.

Responses of the liver to chronic injury include inflammation, regeneration and fibrosis, which finally lead to cirrhosis. The cause of liver cirrhosis appears to be impaired proliferative capability of hepatocytes caused by continuous hepatic damage, and subsequent accumulation of extracellular matrix produced by hepatic stellate cells (HSCs). Epidermal growth factor (EGF) and transforming growth factor-beta1 (TGF-beta1) play a crucial role in hepatocyte proliferation and hepatofibrogenesis, respectively. However, sequential analyses of the intrahepatic expression of EGF and TGF-beta1 in the course of cirrhosis development have not been examined fully. In the present study, liver cirrhosis was produced in rats by intraperitoneal administration of dimethylnitrosamine (DMN), and intrahepatic mRNA expression levels of proliferating cell nuclear antigen (PCNA), EGF and TGF-beta1 were quantitatively estimated by a real-time reverse transcription-polymerase chain reaction method. Histological and semiquantitative densitometric examination of liver sections revealed that the accumulation of extracellular matrix components was increased according to the period of DMN treatment. Histological examination of liver sections of rats treated with DMN for 4 and 6 weeks revealed pre-cirrhosis and cirrhosis, respectively. Intrahepatic mRNA expression levels of PCNA and EGF correlated well. Expression levels of both molecules were increased significantly during the course of cirrhosis development, but decreased significantly at the time of complete cirrhosis manifestation. In contrast, intrahepatic TGF-beta1 expression was increased significantly according to the period of DMN treatment, and reached a peak at the time of cirrhosis manifestation. These results suggest that proliferative capability of hepatocytes was impaired by continuous liver damage due, in part, to the decrease of a hepatocyte mitogen EGF, and that increased intrahepatic TGF-beta1 activated HSCs to retrieve space lost by hepatocyte destruction, resulting in complete cirrhosis manifestation.

Nine-week detection of six genotoxic lung carcinogens using the rasH2/BHT mouse model.

A 9-week in vivo rasH2/butylhydroxytoluene (BHT) model for the detection of genotoxic lung carcinogens was validated, using six potent positive test compounds, dimethylnitrosamine (DMN; 15 mg/kg, i.p.), diethylnitrosamine (DEN; 100 mg/kg, i.p.), ethylnitrosourea (ENU; 120 mg/kg, i.p.), 3-methylcholanthrene (MC; 100 mg/kg, i.p.), 7,12-dimethylbenz(a)anthracene (DMBA; 5 mg/kg, i.g.) and benzo(a)pyrene (B(a)P; 80 mg/kg, i.p.), each given to rasH2 mice of both genders by single administration for initiation followed by promoter BHT treatment. Statistically significant increase in the incidence and multiplicity of lung tumors was observed in rasH2 mice treated with BHT following exposure to all of the carcinogens tested. The data overall suggest the rasH2/BHT model to be a powerful screening tool for genotoxic lung carcinogens.

Rat bladder cell-mediated mutagenesis od Chinese hamster V79 cells and metabolism of benzo[a]pyrene.

Primary rat bladder epithelial cells were cocultivated with Chinese hamster V79 cells in the presence of carcinogens, and the induction of 6-thioguanine resistance in the V79 cells was used as a marker of cell-mediated mutagenesis. The carcinogens dimethylnitrosamine, 7,12-dimethylbenz[a]anthracene, and benzo[a]pyrene (BP) were mutagenic to V79 cells in the presence of bladder cells but not in their absence. Analysis of BP metabolites formed by bladder cells indicated that 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene, 9,10-dihydro-9,10-dihydroxybenzo[a]pyrene, benzo[a]pyrene-3,6-quinone, and 9-hydroxybenzo[a]pyrene were the major organic-soluble metabolites formed. Glucuronide and sulfate conjugates of BP metabolites were also produced by bladder cells. Mutagenesis data from the rat bladder system and previous data from rat liver and lung cell-mediated mutagenesis systems indicate that the cell-mediated mutagenesis approach may provide a useful approach for studying the organotropic effect of chemical carcinogens. Furthermore, the finding that rat bladder epithelium can metabolize some carcinogens offers new possibilities for the mechanism of initiation of bladder cancer.

Morphologic character of transforming renal cell cultures derived from Wistar rats given dimethylnitrosamine.

The morphologic character of kidney cells during serial subculture following isolation from Wistar rats treated several hours to 1 week previously with a carcinogenic dose of dimethylnitrosamine (DMN; 60 mg/kg body weight following protein deprivation) was compared with the appearance of cultures derived from normal control rats. Apart from early signs of cell toxicity, cultures from DMN-treated rats appeared similar to those from untreated rats for the first four passages. Control cells underwent senescence usually by subculture 4, whereas the test cultures survived to express morphologic transformation (usually at subculture 5) as dense macroscopic colonies of piled up cells. In 18 of the 20 test cultures, the cell populations that persisted in continuous culture following expression of morphologic transformation were exclusively mesenchymal, closely resembling DMN-induced renal mesenchymal tumor cells in continuous culture. In the remaining 2 test cultures from DMN-treated rats, a persisting population of abnormal epithelium was present in addition to morphologically transformed mesenchymal cells. The occurrence of populations of altered mesenchymal and epithelial cells characterized by prolonged survival in vitro following isolation from rats shortly after treatment with a carcinogenic dose of DMN was believed to be related to the long-term induction in the rat kidney of a high incidence of mesenchymal tumors and a lower incidence of cortical epithelial tumors by the same dose schedule.

Alkylation of rat liver DNA by dimethylnitrosamine: effect of dosage on O6-methylguanine levels.

Alkylation of liver DNA was studied following administration to Sprague-Dawley rats of doses of dimethylnitrosamine (DMN) varying from 0.25 to 20 mg/kg body weight. Measurements were made of the amounts of O6-methylguanine and 7-methylguanine present in liver DNA at 4 and 24 hours after treatment with the carcinogen. There was a linear relationship between 7-methylguanine levels and dose of the nitrosamine at both of these times. In contrast, the corresponding levels of O6-methylguanine were not directly proportional to dosage but were less than expected, particularly at low doses below 2.5 mg/kg. This discrepancy was significant at 4 hours, but was even more marked at 24 hours. Only doses above 4 mg/kg at the 4-hour time point gave rise to a 0.11 ratio of alkylation of guanine at the O6-position to that at the 7-position. This ratio was that expected for the initial interaction of the alkylating species derived from DMN with DNA. Evidence was obtained to support the hypothesis that these results were due to an enzymatic removal of O6-methylguanine from liver DNA, which occurred much more efficiently at lower initial levels of alkylation. Repeated daily injections of DMN up to 11 days alos gave rise to O6-methylguanine levels that were not proportional to dosage but were relatively greater at higher dose levels. The significance of these findings in the induction of liver cancer by feeding or repeated injection of DMN was explored.

DNA alkylation and tumor induction in regenerating rat liver after cell cycle-related continuous N-nitrosodimethylamine infusion.

Synchronized regenerating rat liver after partial hepatectomy was used to study cell cycle-related DNA base alkylation and liver carcinogenesis. A continuous iv infusion of [14C]N-nitrosodimethylamine (DMN) at a dose of 0.5 mg/kg/hour was given to inbred male Wistar Af/Han rats over a period of 8 hours either during the G1 phase, hydroxyurea-synchronized DNA synthesis, or the G2+M-phase of regenerating liver or to untreated rats (G0-phase liver--carcinogen dose, 1.5 mg/kg/hour). Two hours after the end of the infusion, the amount of 7-methylguanine was highest in the G0-phase liver, with a decrease in the G1 phase, the S-phase, and the G2+M-phase. After continuous DMN exposure, the O6-methylguanine:7-methylguanine ratio was lower in the S-phase and G2+M-phase livers than in the G0-phase and G1-phase livers, indicating an increased O6-methylguanine repair during DNA synthesis and the G2+M-phase. Liver tumors in rats treated by continuous DMN infusion either during the G0 phase or the S-phase developed only after carcinogen exposure during DNA synthesis.

Complement synthesis by guinea pig peritoneal macrophages: failure to detect chemical carcinogens.

In vitro production of the second and fourth components of complement (C2 and C4, respectively) by peritoneal macrophages from noninbred Hartley guinea pigs was tested after the animals had been inoculated with known carcinogens. The system demonstrated the capacity of N-nitrosodimethylamine to decrease C2 and C4 production. However, a similar decrease in C2 and C4 production was seen with only CCl4, 1 of the 10 chemical carcinogens studied. This system had little usefulness as a short-term screening procedure for the detection of carcinogenicity. The effect of the carcinogens on several other functions of peritoneal macrophages was also determined. The number of peritoneal exudate cells (PEC) was significantly lower in carcinogen-inoculated animals than in solvent-inoculated controls for three carcinogens: BeSO4, P < 0.005; CHCl3, P < 0.025; and CCl4, P < 0.01. However, the capacity of the PEC to adhere to plastic was decreased by only CHCl3 (P < 0.05), and adherent cells from all guinea pigs produced normal amounts of total secreted protein.