Several members of the 7-chloro-2,3-dihydro-2-[1-(pyridinyl)alkyl]-pyridazino[4,5-b]quinoline-1,4,10(5H)-triones (2) have been identified as being potent and selective NMDA glycine-site antagonists. Increasing size of the alkyl substituent on the alpha-carbon led to a progressive decrease in binding affinity. Some of these analogues possess improved drug-like properties such as cellular permeability, solubility and oral absorption.

Experiments have demonstrated interlobe differences in the incidence of diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCCA), with a 100% incidence in the left and right median lobes and a 30% incidence in the right anterior lobe 20 weeks after exposure began. These tumor data provide a model to test the hypothesis that chemically induced neoplasia can be qualitatively and quantitatively related to promutagenic DNA damage and concurrent cell replication. Experiments were performed to measure O4-ethyldeoxythymidine (O4-EtdT) (a major pro-mutagenic lesion in hepatic DNA of rats exposed to DEN), N7-ethylguanine, cell replication and hepatocyte initiation using the induction of growth-selected gamma-glutamyl transferase-positive (GGT+) foci in the left and right median and right anterior hepatic lobes following 0, 3, 7, 14 or 28 days of DEN administration. Results demonstrated that O4-EtdT concentrations were consistently higher in the left and right median versus the right anterior hepatic lobes, while cell replication was transiently higher in the right median and right anterior lobes. Likewise, high numbers of GGT+ foci were observed in the left and right median lobes in DEN-exposed rats subjected to a Cayama-Farber growth selection protocol. Following administration of [14C]DEN, the distribution of radioactivity showed a marked left lobe preference in 4-week-old rats that had no prior exposure to DEN and in 8-week-old rats exposed to DEN for 4 weeks. This study suggests that interlobe differences in hepatocyte initiation and the incidence of HCCA may be due in part to differences in cell replication and in DNA alkylation resulting from differential DEN distribution and/or metabolism.

Recent experiments have demonstrated that O6-ethyldeoxyguanosine (O6-EtdG) is efficiently repaired while O4-ethyldeoxythymidine (O4-EtdT) accumulates in hepatocyte DNA of 8-week-old F-344 rats during continuous diethylnitrosamine (DEN) administration. To determine if O4-EtdT accumulation correlates with hepatic initiation, we have quantitated O4-EtdT concentrations, and the incidence of gamma-glutamyl transferase positive (GGT+) foci and hepatocellular carcinoma induced by increasing duration of exposure to DEN in the drinking water (40 p.p.m.). In 8-week-old F-344 rats the number of GGT+ foci increased non-linearly with duration of exposure and reached a maximum of approximately 500 foci/cm3 after 10 weeks. Administration of DEN to 8-week-old F-344 rats for 6 weeks followed by a 15-week administration of 0.05% phenobarbital (PB) in the diet did not result in the induction of hepatocellular carcinoma. Exposure of 4-week-old F-344 rats to DEN for up to 10 weeks produced an O4-EtdT steady-state concentration (approximately 7-10 X 10(-6) mol O4-EtdT/mol dT) similar to that previously observed after administration of DEN to 8-week-old F-344 rats. However, the maximal concentration of O4-EtdT was detectable after a shorter period of DEN administration in the younger rats. The incidence of GGT+ foci also increased more rapidly in 4-week-old rats, but again plateaued at approximately 500 foci/cm3 after 4, 6 or 8 weeks of DEN administration. A 100% incidence of hepatocellular carcinoma occurred in 4-week-old rats administered DEN for 6, 8 or 10 weeks, followed by promotion with 0.05% PB in the diet until week 22 of the study. Lower incidences of hepato-cellular carcinoma (89 and 6%) were observed following PB-promotion of rats administered DEN for 4 and 2 weeks, respectively. The influence of age on DEN-induced hepatic initiation was examined further by quantitating GGT+ foci induced by 4 weeks of DEN administration in groups of rats which were 4-14 weeks old at the start of the carcinogen exposure. The results demonstrated that the younger rats were 15-fold more susceptible than the older rats to the initiating effects of DEN. This growth-dependent effect on hepatic initiation in the presence of nearly equivalent amounts of pro-mutagenic DNA damage further implicates the necessity of replication for hepatic initiation.

An impressive array of evidence has been obtained during the past decade establishing correlations between specific DNA adducts and carcinogenesis. Many of the studies utilized organ specific differences in carcinogenesis to establish the correlations. More recently, we have investigated similar relationships between target and nontarget cell populations within the liver. Chronic exposure to methylating hepatocarcinogens predominantly induces hemangiosarcomas, whereas exposure to ethylating agents causes hepatocellular carcinomas. This cell specificity in carcinogenesis correlates well with the presence of promutagenic DNA adducts. In the case of methylating agents, the nonparenchymal cells accumulate O6-methylguanine whereas the hepatocytes do not. Exposure to ethylating agents leads to accumulation of O4-ethyldeoxythymidine, but not O6-ethyldeoxyguanosine in hepatocytes. These differences reflect the ability of the two cell populations to repair O6-alkylguanine and the extent of purine and pyrimidine alkylation with methylating and ethylating agents. Hepatocytes of rats exposed to diethylnitrosamine for 28 days have four to five times more promutagenic DNA adducts (O6-alkyldeoxyguanosine and O4-alkyldeoxythymidine) than hepatocytes of rats exposed to nearly equimolar doses of dimethylhydrazine. Both O6-methylguanine and O4-methyldeoxythymidine are rapidly repaired by rat hepatocytes, while only O6-ethyldeoxyguanosine is rapidly repaired. Studies comparing the relationship between the induction of gamma-glutamyl transpeptidase-positive foci, hepatocellular carcinoma and promutagenic lesions such as O4-ethyldeoxythymidine will be useful in understanding associations between the molecular dosimetry of DNA adducts, initiation, and progression of hepatocarcinogenesis.

The mononitrotoluenes are important industrial chemicals which display isomeric specificity in their ability to induce hepatic DNA excision repair in Fischer-344 rats. Covalent binding of the structurally related hepatocarcinogen, 2,6-dinitrotoluene, to hepatic DNA is markedly decreased by prior administration of the sulfotransferase inhibitors pentachlorophenol (PCP) and 2,6-dichloro-4-nitrophenol (DCNP). The objectives of this study were to determine whether hepatic macromolecular covalent binding of the mononitrotoluene isomers differed and to determine whether covalent binding of the mononitrotoluenes to hepatic DNA in vivo was decreased by inhibitors of sulfotransferase. Male Fischer-344 rats were given a single oral dose of [ring-U-14C]-2-, 3- or 4-nitrotoluene (2-, 3- or 4-NT) and killed at various times thereafter. Livers were removed and analyzed for total and covalently bound radiolabel. Maximal concentrations of total radiolabel were observed between 3 and 12 h after the dose, and there were no large differences among the 3 isomers in peak concentrations achieved. Covalent binding to hepatic macromolecules was maximal 12 h after administration for all three isomers. Thereafter, concentrations of covalently bound 2-NT-derived material were always 2-6 times higher than those of 3- or 4-NT-derived material. When DNA was isolated from livers of rats given the mononitrotoluenes 12 h previously, only 2-NT was observed to covalently bind at concentrations above the limits of detection of the assay. The covalent binding of 2-NT, but not that of 3- or 4-NT, to both total hepatic macromolecules and DNA was markedly decreased by prior administration of either PCP or DCNP. Covalent binding to hepatic DNA was decreased by greater than 96%. The results of this study correlate well with studies which have demonstrated that 2-NT, but not 3- or 4-NT, induces DNA excision repair. Furthermore, they suggest that 2-NT, like the hepatocarcinogen 2,6-dinitrotoluene, requires the action of sulfotransferase for its conversion to a species capable of covalently binding to hepatic DNA.

The objectives of these experiments were to determine N-7-methylguanine (m7Gua) and O6-methylguanine (O6mGua) concentrations in DNA, [3H]thymidine uptake into DNA, and O6mGua-DNA methyltransferase activity in hepatocytes of F-344 rats and C3H and C57BL mice exposed to 0, 10, 30, or 100 ppm dimethylnitrosamine (DMN) ad libitum in their drinking water for 16 days. The 100-ppm DMN exposure regimen was lethal to the C3H mice. Using water consumption and body weight to surface area conversions, these exposures averaged 5, 13, and 27 mg/sq m/day for F-344 rats, 6, 16, and 31 mg/sq m/day for C57BL mice, and 6 and 16 mg/sq m/day for C3H mice. Over a 5-fold range of DMN exposure, m7Gua concentrations in DNA of rat hepatocytes increased 9-fold, while O6mGua concentrations increased only 3-fold. In contrast, while m7Gua increased 4-fold, O6mGua increased 14-fold in both strains of mice. O6mGua-DNA methyltransferase activity in rat hepatocytes was increased to 150% that of control values at the low exposure, and to 200% at the intermediate and high exposures of DMN. Methyltransferase activity in both strains of mice decreased with increasing exposure to DMN, such that C3H hepatocytes had only 59 and 20% as much activity as controls, while C57BL hepatocytes had 68, 38, and 14% as much methyltransferase activity. Relative to controls, the only significant increase in [3H]thymidine uptake into DNA of hepatocytes occurred at 30 ppm DMN in C3H mice. We conclude that under conditions of DMN exposure leading to comparable m7Gua and O6mGua concentrations in DNA, O6mGua-DNA methyltransferase activity is enhanced in F-344 rats, but partially depleted in C57BL and C3H mice.

The sulfotransferase inhibitors 2,6-dichloro-4-nitrophenol and pentachlorophenol were used to investigate the role of sulfate ester formation during the in vivo bioactivation of 2,4- and 2,6-dinitrotoluene (DNT). Male F-344 rats were administered one of the sulfotransferase inhibitors (40 mu mol/kg i.p.) 45 min prior to oral administration of 28 mg/kg [ring-14C]-2,4-DNT or [3-3H]-2,6-DNT and killed 12 h later. Pentachlorophenol had no significant effect on the urinary excretion of the benzyl glucuronide or benzoic acid metabolites of 2,6-DNT. The sulfotransferase inhibitors decreased the total hepatic macromolecular covalent binding of 2,4-DNT by 33%, and of 2,6-DNT by 69%. Purification of hepatic DNA by hydroxylapatite chromatography indicated covalent binding of 2,4- and 2,6-DNT at levels of 45 and 94 pmol equivalents/mg DNA, respectively. The sulfotransferase inhibitors decreased the binding of the hepatocarcinogen 2,6-DNT to hepatic DNA by greater than 95%. 2,6-Dichloro-4-nitrophenol decreased the binding of 2,4-DNT to DNA by greater than 84% while the decrease due to pentachlorophenol was 33%. These results suggest that sulfation is important in the biotransformation of 2,4- and 2,6-DNT to reactive metabolites which covalently bind to DNA. 3H2O was detected in the urine of rats administered [3-3H]-2,6-DNT. Pentachlorophenol decreased 3H2O formation to the same extent as it decreased the total hepatic macromolecular covalent binding of 2,6-DNT, suggesting that 3H exchange at the 3 position of 2,6-DNT occurs following sulfate ester formation. These results are consistent with a nitrenium-carbonium ion resonance of the sulfate ester-derived reactive intermediate of 2,6-DNT.

In previous investigations into the mechanisms responsible for cell specificity in hepatocarcinogenesis, we have demonstrated that O6-methylguanine accumulates in the DNA of nonparenchymal cells (NPC) but is efficiently removed from hepatocellular DNA. O6-Alkylguanine may, therefore, be an important promutagenic lesion responsible for the induction of hepatic angiosarcomas after exposure to methylating agents, but other promutagenic DNA alkylation products--i.e., O4-alkylthymine--may be responsible for the initiation of hepatocellular carcinomas. F-344 male rats were provided drinking water containing diethylnitrosamine (DEN) at 40 ppm for 0, 2, 4, 8, 16, 28, 49, or 77 days, a regimen that selectively causes hepatocellular carcinomas. Hepatocytes and NPC were isolated by using low-speed differential centrifugation. DNA was purified by hydroxyapatite chromatography and hydrolyzed enzymatically, and O4-ethyldeoxythymidine (O4-EtdThd) and O6-ethyldeoxyguanosine (O6-EtdGuo) of hepatocyte and NPC DNA were quantitated by competitive radioimmunoassay using high-affinity monoclonal antibodies. O4-EtdThd accumulated in hepatocyte DNA during the first 28 days of DEN exposure, approximating a steady state at an O4-EtdThd-to-deoxythymidine molar ratio of approximately equal to 1 X 10(-5). This O4-EtdThd concentration was maintained from 28 to 77 days of DEN exposure. In contrast, O6-EtdGuo did not accumulate in hepatocyte DNA, its greatest concentration O6-EtdGuo-to-deoxyguanosine ratio (approximately equal to 3.7 X 10(-7) ) being detected after 2 days of exposure to DEN. O6-EtdGuo concentrations in hepatocyte DNA decreased with duration of exposure to DEN to an O6-EtdGuo-to-deoxyguanosine ratio of less than 2 X 10(-7) from 28 to 77 days. The data indicate that O4-EtdThd disappears from the DNA of hepatocytes less than 1/200th as fast as O6-EtdGuo. DNA from NPC contained approximately half as much O4-EtdThd as hepatocytes did, but greater than or equal to 2.5 times more O6-EtdGuo.

Lorsque des souches de souris ayant une forte (C3H) et une faible (C57BL) incidence de carcinome hepatocellulaire spontane sont comparees aucune difference dans l'activite de l'O 6 AGAAP n'a pu etre mise en evidence dans les hepatocytes des souris de controle et des souris exposees a la dimethylnitrosamine. Donc l'activite de la O 6 AGAAP n'est pas un facteur dans le developpement des carcinomes spontanes hepatocellulaires de la souris

The in vivo covalent binding of the hepatocarcinogen thioacetamide to rat liver protein has been examined. Following administration of 3H- or 14C-labeled thioacetamide, the modified amino acids present in the hepatic cytosolic proteins were isolated by enzymatic digestion and ion-exchange chromatography. Approximately 70% of the radioactivity covalently bound to cytosolic protein was recovered in a compound which upon acid hydrolysis yielded lysine and radiolabeled acetate. Additional studies indicated the structure of this adduct was N-epsilon-acetyllysine.